N-acetylcarboxymethylchitosan derivatives and process for preparation thereof

ABSTRACT

N-acetylcarboxymethylchitosan derivatives are provided by the present invention. They are represented by the following formula (I): ##STR1## wherein R 1  and R 2  individually mean H or carboxymethyl group; P denotes a group R 3  CO--, a group R 4  NH-- or a group R 5  O-- with assuming that R 3  COOH denotes a compound having carboxyl group, R 4  NH 2  denotes a compound having amino group and R 5  OH denotes an alcohol compound; Q stands for H or a group --OH; X represents a peptide chain containing same or different, one to ten amino acids; a 1  and a 2  individually represent zero or a positive integer, provided that both of a 1  and a 2  are not zero at the same time, and b stands for a positive integer; and having the following characteristic values (1)-(4): 
     
         ______________________________________                                    
 
    
     (1)   carboxymethylation degree:                                          
                         0.5-1.2                                          
(2)   molecular weight (as measured                                       
                          3,000-300,000                                   
      by gel filtration method):                                          
(3)   a/(a + b):         0.01-1                                           
      [provided that a = a 1  + a 2  ]                              
(4)   P/X ratio (molar ratio):                                            
                         0.1-1                                            
______________________________________                                    
 
     P in the formula (I) can be the residue of a pharmaceutical compound. In this case, the substances of the formula (I) are derivatives of the pharmaceutical compound, which derivatives have been improved in the properties such as organotropism in vivo. 
     P in the formula (I) can also be protective groups. In this case, removal of the protective groups from the substances of the formula (I) by deprotection makes it possible to provide such N-acetylcarboxymethylchitosan derivative which is useful as carrier for pharmaceutical compounds and which has the terminals of the so deprotected peptide chains capable of being linked with a pharmaceutical compound. 
     There are further obtained such derivatives of the substances of the formula (I), in which derivatives some of the N-acetylcarboxymethylglucosamine units have been replaced by units having a high solubility in water.

TECHNICAL FIELD

This invention relates to novel N-acetylcarboxymethylchitosanderivatives or N-acetylated carboxymethylchitosan derivatives andprocesses for the preparation thereof. More specifically, the presentinvention is concerned with an in vivo targeting technique for medicalor pharmaceutical compounds, and thus concerned with novelN-acetylcarboxymethylchitosan derivatives which are useful aspolysaccharide-type of high-molecular carriers utilizable for enhancingthe stability of pharmaceutical compounds in blood, the organotropism ofpharmaceutical compound and the biodegradability of medicinal productprepared. This invention also pertains to novelN-acetylcarboxymethylchitosan derivatives in the form of complexes aslinked to the pharmaceutical compounds.

BACKGROUND ART

Use of water-soluble, high-molecular substances as the carriers fordrugs, namely, pharmaceutical compounds has been attempted to dateespecially in the field of pharmaceutical preparations, and a number ofrelated techniques have been provided. These water-soluble,high-molecular substances as the drug carriers are already known widely.In many instances, a cellulose derivative such ascarboxymethylcellulose, hydroxypropylcellulose orhydroxypropylmethylcellulose is used. However, these conventionaltechniques are concerned with so-called sustained release of drugs andhave not been developed to techniques for delivery of a drug to a tissuewhere the drug is required, when necessary, only in an amount as needed.It is the current situation that no satisfactory technique has beendeveloped yet for using a water-soluble, high-molecular substance as acarrier for delivery of the drug. For example, there are thebelow-described publications 1) and 2). The publication 1) discloses useof carboxymethylchitin as a fine particulate carrier of the implantedtype, whereas the publication 2) discloses use of carboxylated dextranas a carrier for the formation of its complex with a drug.

1) WATANABE, K. et al., "Chem. Pharm. Bull.," 38, 506-509, (1990), and

2) SEZAKI, Hitoshi, "Yakugaku Zasshi,: 109, 611-621, (1989)

Although the technique disclosed in the publication 1) perceived thegelling ability and the in vivo biodegradability of carboxymethylchitinand makes use of these properties, carboxymethylchitin is of the typewhich is implanted to a particular local site in the body so that thistechnique does not go beyond the boundary of the controlled release of adrug and is not expected to enhance the organotropism of a drug to atarget cancer tissue or a target organ. On the other hand, the drugcomplexes disclosed in the publication 2) suggests the possibility ofexcellent drug-delivery, but functional groups of said carboxylateddextran which are usable for the molecular modification ofdrug-complexes to facilitate the delivery of drugs are limited toalcoholic hydroxyl groups.

Reflecting the development of an increasing number of the anticanceragents, brain disease-curing drugs and the like, there is an outstandingneed for the urgent establishment of targeting technology for thesedrugs with using a water-soluble, high-molecular substance.Nevertheless, the prior art techniques have not brought about any fullysatisfactory solution.

To complete a targeting technique for drugs, namely, pharmaceuticalcompounds, by using a water-soluble, high-molecular carrier, it isnecessary as prerequisites therefor, firstly that a complex of carrierwith a drug remains stable in blood after its administration byintravenous injection until its arrival at a target organ, in otherwords, the drug can be maintained at a necessary concentration in blood,secondly that the carrier is subjected to gradual degradation in thebody and consequently the carrier does not remain for a long time in thehuman body, and thirdly that the carrier as linked to the drug in theform of the complex exhibits by itself the tendency of an organotropismin vivo. Therefore a carrier must be provided, first of all, as onebeing capable of meeting these requirements all together.

DISCLOSURE OF THE INVENTION

With a view toward overcoming such problems, the present inventors haveproceeded with a variety of investigations. As a result, the presentinventors have now succeeded in synthesizing as novel substances certainN-acetylcarboxymethylchitosan derivatives, namely N-acetylatedcarboxymethylchitosan derivatives, by introducing an N-peptide chain andN-acetyl group into amino groups of such a depolymerisedcarboxymethylchitosan, which has been obtained by subjecting theaforementioned carboxymethylchitin to enzymatic treatment and alkalitreatment. In addition, we, the present inventors, have unexpectedlyfound that these N-acetylcarboxymethylchitosan derivatives are useful ascarriers capable of satisfying the different requirements describedabove and also that their use can achieve the above objects. It has alsobeen revealed that the selection of suitable peptide chains as saidN-peptide chain to be introduced will enable to broaden the range ofapplicable pharmaceutical compounds to the wide range of compoundshaving carboxyl group, compounds having amino group, and alcoholiccompounds and, moreover, can provide the resulting complexes with atendency of the organotropism in vivo to a particular organ. Thisinvention has been completed based on these findings.

The present invention will hereinafter be described in detail.

Novel substances according to the present invention areN-acetylcarboxymethylchitosan derivatives represented by a generalformula (I) given below. These novel derivatives are characterized asone of their features by that the hydrogen atoms of some of the6-hydroxyl and/or 3-hydroxyl groups of glucosamine, which is theconstituent sugar unit of the chitosan of said derivatives, have beenreplaced by carboxymethyl groups; the hydrogen atoms of the 2-aminogroups present in some of the sugar units have been replaced by acetylgroups; and the hydrogen atoms of the 2-amino groups present in othersome of the sugar units have been replaced by peptide chains or aminoacids.

A novel N-acetylcarboxymethylchitosan derivative according to the firstaspect of the present invention is represented by the following generalformula (I): ##STR2##

In the formula (I), R₁ and R₂ individually mean H or carboxymethyl groupwith the proviso that they do not stand for H at the same time.

Further, in the above formula, P denotes a group R₃ CO--, a group R₄NH-- or a group R₅ O--. Here, in an instance, P may be linked to anN-terminal amino acid of an N-peptide chain which is the peptideattached to the 2-amino group of the glucosamine moiety and isrepresented by X. In this case, P means the group R₃ CO--. In anotherinstance, P may be linked to a C-terminal amino acid of said N-peptidechain as represented by X, and in this instance P may mean the group R₄NH-- or the group R₅ O--. In the above, it is assumed that R₃ COOHdenotes a compound having carboxyl group, that is, a compound ofcarboxylic acid-type, R₄ NH₂ denotes a compound having amino group andR₅ OH denotes an alcohol compound.

Described specifically, in many instances, the R₃ CO-- group can be aprotective group for an amino group, or an acyl group originated from apharmaceutical compound of the carboxylic acid-type. Illustrativeexamples of such amino-protecting groups include alkoxycarbonyl groupssuch as tert-butoxycarbonyl, and aralkyloxycarbonyl groups such asp-methoxybenzyloxycarbonyl. As an alternative, the R₃ CO-- group canalso be such a group that its corresponding R₃ COOH denotes apharmaceutical or drug compound of the carboxylic acid-type, forexample, methotrexate.

Examples of the R₅ O-- group include lower alkoxyl groups useful asprotective groups for carbonyl group, for example, t-butyloxy; andaralkyloxy groups, for example, benzyloxy. As an alternative, the R₅ O--group can be such a residue which is formed by removing H from thealcoholic hydroxyl group of an alcohol-type pharmaceutical compound R₅OH.

The R₄ NH-- group can be a protective group of the amido-type forprotecting carboxyl group, for example, a lower alkylimino group such asmethylimino. As an alternative, the R₄ NH-- group can be such a groupthat its corresponding R₄ NH₂ represents a pharmaceutical compound ofthe type of compounds having amino group(s), for example, daunorubicinor triprolidine.

Q stands for H or a group --OH. Q may be bonded to the N-terminal aminoacid of the N-peptide chain as represented by X. In this case, Q meansH. In another case, Q may alternatively be bonded to the C-terminalamino acid of the N-peptide chain as represented by X, wherein Q meansthe group --OH.

Further, X in the formula (I) represents a peptide chain containing sameor different, one to ten amino acid(s). Here, it is to be noted that asingle amino acid is also interpreted as being embraced by the peptidesin this specification. This "peptide chain" should be interpreted asembracing not only such peptide chains composed of amino acids alone,but also such peptide chains comprising amino acids and containing oneor more compounds different from the amino acids in part(s) of thepeptide chain. In the latter case, the different compound(s) may be adibasic acid, especially a dibasic carboxylic acid such as succinic acidwhich can be interposed and connected to an intermediate or terminalamino acid(s) in the peptide chain so that peptide chain(s) containingsame or different amino acids is or are constructed as a wholeconsequence.

In addition, the N-acetylcarboxymethylchitosan derivative represented bythe formula (I) includes its salts, especially alkali metal salts at thecarboxymethyl group of said derivative, for example, the sodium,potassium and ammonium salts.

In the derivative having the formula (I) according to the first aspectof this invention, the number of amino acids forming the peptide chain Xmay be generally 1-4 in account of release of the drug compound andantigenicity of the derivative, with 3-4 amino acids being particularlypreferred. The followings are examples of the moiety XP in such caseswhere the peptide chain X present in XP in the formula (I) represents apeptide chain composed of 1-4 amino acids alone and P is linked to theN-terminal amino acid of the peptide chain. In such cases, the peptidebond existing in the peptide chain X is in the form of --NHCO-- asviewed from the side of the chitosan unit.

P-Phe-Phe-Gly-,

P-Gly-Phe-Gly-Gly-,

P-Phe-Gly-Phe-Gly-,

P-Gly-Phe-Gly-Phe-,

P-Gly-Gly-Gly-, and

P-Ala-Gly-Gly-Gly-.

The followings are examples of the moiety XP in such instances where thepeptide chain X present in XP represents a peptide chain containing adibasic acid and 1-4 amino acids and P is linked to the C-terminal aminoacid of the peptide chain. In such instances, the peptide bond existingin the peptide chain X is in the form of --CONH-- as viewed from theside of the chitosan unit.

-Suc-Ala-Ala-Ala-P, and

- Suc-Ala-Ala-Val-Ala-P

in which Suc represents the residue of succinic acid. Further, otherillustrative examples of the amino acid sequences usable for the peptidechain X in accordance with this invention include:

(i) H-Gly-Gly-Gly-Val-Ala-OH, -Gly-Gly-Gly-Leu-Ala-,-Gly-Gly-Phe-Leu-Gly-, -Gly-Gly-Phe-Try-Ala-, -Gly-Gly-Gly-Gly-Gly-,

(ii) -Gly-Gly-Gly-Phe-Leu-Gly-, -Gly-Gly-Gly-Gly-Leu-Ala-,-Gly-Gly-Gly-Gly-Gly-Gly,

(iii) -Gly-Gly-Gly-Gly-Phe-Leu-Gly-, -(Gly)₇ -, -(Gly)₅ -Lue-Ala-,

(iv) --(Gly)₅ -Phe-Leu-Gly, -(Gly)₈ -,

(v) -(Gly)₉ -, and

(vi) -(Gly)₁₀ -.

The followings are illustrative examples of parent drug compounds fromwhich the residue (P-) of the pharmaceutical compound usable in thisinvention can be derived:

Methotrexate, levadopa, bumetanide, furosemide, dinoprost, daunorubicin,doxorubicin, mitomycin C, triprolidine, acriflavinum, etc.

Further, the compound of the formula (I) according to the first aspectof this invention is characteristically specified by itscarboxymethylation degree, molecular weight (as measured by gelfiltration method), values of the ratio a/(a+b) and ratio P/X(molarratio).

The carboxymethylation degree is determined by colloidal titration oralkalimetry of the compound of the formula (I) in this invention. Withrespect to the method for colloidal titration, reference is made to thefollowing publication 3):

3) OKIMASU, Satoru, "Journal of the Society of Agricultural Chemistry",32, 303-308, (1958)

In the present invention, the molecular weight of the compound of theformula (I) according to this invention is determined by gel filtrationmethod with using a dextran as standard substance. In Examples givenhereinafter, the molecular weight of the compound of the formula (I) andothers was measured using a column of "TSK-gel G4000PW_(XL) " as elutedwith aqueous 0.1M sodium chloride solution fed at a flow rate of 0.8ml/minute and at a column temperature of 40° C. while detecting by meansof differential refractometer.

The above-mentioned value of a/(a+b) is a value which may be called,e.g., "peptide-introducing degree". In this value, a represents the sumof the number a₁ of sugar units as substituted by --XP plus the numbera₂ of sugar units as substituted by --XQ, and b stands for the number ofacetyl-substituted sugar units. Each of the sugar units of the compoundof the formula (I) is substituted either by --XP, or by --XQ or by theacetyl group so that a+b indicates the total number of the sugar unitsin the molecule of said compound.

Incidentally, a/(a+b) is determined by the following equation (1):##EQU1## wherein A means the content (wt. %) of P in the compound of theformula (I); M_(m) represents the molecular weight of P; M_(s) denotesthe average molecular weight of the sugar units ofN-acetylcarboxymethylchitosan; and C stands for the content (wt. %) of X(when a₂ =0) in the compound of formula (I). C is calculated inaccordance with the following equation (2):

    C=B(100-A)×10.sup.-2                                 ( 2)

wherein B represents the content (wt. %) of X (when a₁ =0) in thecompound of formula (I). B can be determined by measuring the content ofthe peptide, for example, after the introduction of peptide but beforethe introduction of P or after removing P from the compound of theformula (I). If the amino acids in X show a characteristic absorption,this can be used to determine B directly by absorptionspectrophotometry. If the amino acid(s) in X does not have anycharacteristic absorption, however, B can be determined in accordancewith the following equation (3). ##EQU2## wherein D represents thecontent (wt. %) of XP (when a₂ =0) in the compound of formula (I). If Phas a characteristic absorption, this can be used to determine D byabsorption spectrophotometry. Further, r indicates a value determined bythe following equation (4):

    r=M.sub.PX /M.sub.X                                        ( 4)

wherein M_(PX) and M_(X) represent the molecular weights of XP and X,respectively.

Next, the molar ratio P/X is a value which may be called, for example, adegree of introducing pharmaceutical compound (P) into the peptide (ortermed as degree of substitution with P), and which is corresponding toa value of a₁ /(a₁ +a₂). The molar ratio P/X can be determined inaccordance with the following equation (5): ##EQU3##

In the compound of formula (I) according to the first aspect of thisinvention, the carboxymethylation degree, molecular weight (as measuredby gel filtration method), the a/(a+b) value and the P/X value, whichare calculated as described above, are in the ranges of 0.5-1.2,3,000-300,000, 0.01-1 and 0.1-1, respectively.

In the present specification, the formula (I) does not indicate anythingbeyond that, in the molecule of the compound represented by the formula(I), the number of sugar units as substituted by --XP (i.e., glucosaminesugar units) is a₁, the number of sugar units as substituted by --XQ isa₂, and the number of sugar units as substituted by --COCH₃ is b. Theformula (I) indicate neither that the substituted sugar units of each ofthe aforesaid types are successively bonded together as many as theircorresponding number a₁, a₂ or b, nor that the sugar units of theaforesaid three types are bonded together in the sequence or order asexhibited in the formula (I).

In addition, a partially N-acetylated carboxymethylchitosan derivativewhich is lacking some of the N-acetyl groups originally present in thecompound of formula (I) according to the first aspect of this inventionis also a novel substance and has a utility, as will be describedsubsequently.

In a second aspect of this invention, therefore, there is thus provideda partially N-acetylated carboxymethylchitosan derivative represented bythe following formula (II): ##STR3## In the formula (II), R₁, R₂ and Xhave the same meanings as defined in the formula (I) but P is somewhatbroader in its meaning than its definition as given in the formula (I).P denotes H, a group --OH, a group R₃ CO--, a group R₄ NH-- or a groupR₅ O--. P may be linked to the N-terminal amino acid of the peptidechain X, in some instances. P stands for H or the group R₃ CO-- in thiscase. P may also be linked to the C-terminal amino acid of the peptidechain X, in some instances. P stands for the group --OH, the group R₄NH-- or the group R₅ O-- in this case. a, b₁ and b₂ individuallyrepresent a positive integer.

Further, the compound of the formula (II) can also be characterized byits carboxymethylation degree and molecular weight (as measured by gelfiltration method) as well as by the a/(a+b) value and the P/X molarratio which are determined by the same methods as those referred toabove with respect to the formula (I). However, b in the value a/(a+b)in the compound of formula. (II) is somewhat different in its meaningfrom b for the compound of the formula (I), and it represents the sum ofthe number b₁ of acetyl-substituted sugar units plus the number b₂ ofsugar units in each of which the 2-amino group remains free withoutbeing substituted. These characteristic values of the compound of theformula (II) are in the same ranges as those of the compound having theformula (I).

In the present specification, the formula (II) does not indicateanything beyond that, in the molecule of the compound represented by theformula (II), the number of sugar units as substituted by --XP is a, thenumber of sugar units as substituted by --COCH₃ is b₁, and the number ofsugar units having the 2-amino groups remaining free and unsubstitutedis b₂. The formula (II) indicate neither that the sugar units of each ofthe aforesaid three types are successively bonded together as many astheir corresponding number a, b₁ or b₂ , nor that the sugar units of thethree types are bonded together in the sequence or order as shown in theformula (II).

The compound of the formula (II) may be an intermediate useful forsynthetizing and preparing the substance of the formula (I) and,accordingly, has the industrial applicability and principal structure ofthe compound same as the compound of the formula (I).

For example, the compound of the formula (II) where P is the residue R₃CO--, R₄ NH-- or R₅ O-- derived from a pharmaceutical compound is ableto afford the compound of the formula (I) in the form of the complexlinked to the pharmaceutical compound, when the amino groups of theformer compound (II) as such are acetylated with acetic anhydride oranother suitable acetylating agent such as acetyl chloride in anappropriate solvent, for example, pyridine. The compound of the formula(I) according to the present invention can also be produced if R₃ COOH,R₄ NH₂ or R₅ OH useful as a pharmaceutical compound is either made to belinked to the compound of the formula (II) after removal of theprotective groups from the compound of the formula (II) where Ps havebeen the protective groups, or if such R₃ COOH, R₄ NH₂ or R₅ OH is madeto be linked directly to the compound of formula (II) where Ps are Hs or--OH, followed by acetylation of the remaining amino groups as describedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings,

FIG. 1 shows an ultraviolet absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-Boc complex as obtained inExample 1 of this invention (at a concentration of the complex of 1900μg/ml in 30% aqueous ethanol as solvent);

FIG. 2 shows a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-Boc complex as obtained inExample 1 of this invention (with detection of the complex being made byultraviolet absorption at 258 nm);

FIG. 3 shows an ultraviolet absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-H complex as obtained inExample 1 of this invention (at a concentration of the complex of 1900μg/ml in aqueous 30% ethanol as solvent);

FIG. 4 shows a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-H complex as obtained inExample 1 of this invention (with detection of the complex being made byultraviolet absorption at 258 nm);

FIG. 5 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-MTX complex obtained as thefinal product in Example 1 of this invention (at a concentration of thecomplex of 100 μg/ml in aqueous 0.1% NaHCO₃ as solvent);

FIG. 6 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-MTX complex obtained as thefinal product in Example 1 of this invention (with detection of thecomplex being made by ultraviolet absorption at 307 nm);

FIG. 7 shows an ultraviolet absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-pMZ complex as obtained inExample 2 of this invention (at a concentration of the complex of 500μg/ml in water as solvent);

FIG. 8 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-pMZ complex as obtained inExample 2 of this invention (with detection of the complex being made byultraviolet absorption at 272 nm);

FIG. 9 shows an ultraviolet absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-H complex as obtained inExample 2 of this invention (at a concentration of the complex of 1000μg/ml in water as solvent);

FIG. 10 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-MTX complex obtained as thefinal product in Example 2 of this invention (at a concentration of thecomplex of 48 μg/ml in aqueous 0.1% NaHCO₃ as solvent);

FIG. 11 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-MTX complex obtained as thefinal product in Example 2 of this invention (with detection of thecomplex being made by ultraviolet absorption at 307 nm);

FIG. 12 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-MTX complex as obtainedin Example 6 of this invention (at a concentration of the complex of 50μg/ml in aqueous 0.1% NaHCO₃ as solvent);

FIG. 13 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-MTX complex as obtainedin Example 6 of this invention (with detection of the complex being madeby ultraviolet absorption at 307 nm);

FIG. 14 shows an ultraviolet absorption spectrum of theN-acetylcarboxymethylchitosan--Suc-Ala-Ala-Ala-p-nitroanilide complexobtained as the final product in Example 7 of this invention (at aconcentration of the complex of 500 μg/ml in water as solvent);

FIG. 15 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Suc-Ala-Ala-Ala-p-nitroanilide complexobtained as the final product in Example 7 of this invention (withdetection of the complex being made by ultraviolet absorption at 315nm);

FIG. 16 is a graph showing time-dependent variations in the percentagesof the residual content of the pharmaceutical compound, methotrexate(MTX), having retained the form of its complex with the chitosan withinplasma (in vitro), which were measured in Experiment 1 of thisinvention;

FIG. 17 is a graph showing how the reaction time of the degradation ofthe complexes of this invention by reacting with an acetate buffer wasco-related to the elution time at which the elution of a peak of thecomplex appeared upon the elution of the reaction mixture at thedifferent time points of the reaction time when the measurement was donein Experiment 2 of this invention;

FIG. 18 is a graph showing time-dependent variations in theconcentration or level of a test sample compound present in blood serum(in vivo) when the measurement was done in Experiment 3 of thisinvention;

FIG. 19 is a graph showing time-dependent variations in theconcentration of a test compound present in the cancer tissue when themeasurement was done in Experiment 3 of this invention;

FIG. 20 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Suc-Gly-Phe-Gly-Lys-MTX complex asobtained in Example 11 of this invention (at a concentration of thecomplex of 490 μg/ml in aqueous 0.1% NaHCO₃ as solvent);

FIG. 21 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Suc-Gly-Phe-Gly-Lys-MTX complex asobtained in Example 11 of this invention (with detection of the complexbeing made by ultraviolet absorption at 307 nm);

FIG. 22 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-MTX complex (with itscarboxymethylation degree of 0.7) as obtained in Example 12 of thisinvention (at a concentration of the complex of 100 μg/ml in aqueous0.1% NaHCO₃ as solvent);

FIG. 23 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Phe-Phe-MTX complex (with itscarboxymethylation degree of 0.7) as obtained in Example 12 of thisinvention (with detection of the complex being made by ultravioletabsorption at 307 nm);

FIG. 24 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--(PEG₁)--Gly-Phe-Phe-H complex as preparedin Example 17 of this invention (with detection of the complex beingmade by ultraviolet absorption at 258 nm);

FIG. 25 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--(PEG₁)--Gly-Phe-Phe--MTX complex obtainedas the final product in Example 17 of this invention (at a concentrationof the complex of 100 μg/ml in aqueous 0.1% NaHCO₃ as solvent);

FIG. 26 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--(PEG₁)--Gly-Phe-Phe--MTX complex obtainedas the final product in Example 17 of this invention (with detection ofthe complex being made by ultraviolet absorption at 307 nm);

FIG. 27 shows an ultraviolet-visible absorption spectrum of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex as convertedinto a polyol derivative which was obtained in Example 22 of the presentinvention (at a concentration of the complex of 40 μg/ml in aqueous 0.1%NaHCO₃ as solvent);

FIG. 28 depicts a GFC elution pattern of theN-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex as convertedinto a polyol derivative which was obtained in Example 22 of thisinvention (with detection of the complex being made by ultravioletabsorption at 307 nm);

FIG. 29 is a graph showing time-dependent variations in theconcentrations of various sample complexes present in the plasma of ratsas measured up to the end of 24 hours after administration of thecomplexes when the measurement was done in Experiment 4 of thisinvention; and

FIG. 30 is a graph showing the distributions of various sample complexeswhich were present in different tissues of bodies of rats, and whichwere expressed as the concentration of the complex in terms of thepercentages on the basis of the dose of each complex.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A description will next be made of the preparation of the compound offormula (I) according to the first aspect of this invention. Although itcan be outlined as will be described below, the present invention is notbe limited to the following preparation method.

Carboxymethylchitin, one of the starting materials for production of thenew substance of this invention, can be easily prepared by reacting, inthe presence of an alkali, monochloroacetic acid with chitin (namely,β-1, 4-poly-N-acetylglucosamine) which is found widely in cruschymatasuch as crabs, shrimps and lobsters, shells of insects, cell walls offungi, and the like. By modifying the reaction conditions,carboxymethylchitin can be obtained with varying carboxymethylationdegrees. Commercial products can also be used.

Next, carboxymethylchitin is reduced in its molecular weight, namely,degraded or depolymerized. The molecular weight of the resultingdepolymerized carboxymethylchitin can be regulated, for example, bycausing egg white lysozyme to act on a commercially availablecarboxymethylchitin under controlled reaction conditions upondepolymerization of the commercially available carboxymethylchitin. Thereaction conditions therefor also can vary depending on thecarboxymethylation degree (ds: carboxymethylation degree per sugarresidue) of carboxymethylchitin to be employed. Where the value of ds is1.0, for example, a depolymerized carboxymethylchitin having a molecularweight reduced to about 1×10⁵ can be obtained by reactingcarboxymethylchitin, whose molecular weight is about 1×10⁶ or so, withabout 1/400 volume of egg white lysozyme at pH 6.0 and 37° C. for about2 hours.

The carboxymethylchitin so depolymerized is further subjected tode-N-acetylation. This can be achieved by alkali treatment. Bydissolving the depolymerized carboxymethylchitin, for example, in 1NNaOH and heating the resulting mixture under reflux at 100° C. forseveral hours to several tens hours, preferably 3-8 hours, the N-acetylgroups are partially removed therefrom so that carboxymethylchitosanhaving some N-acetyl groups still remaining therein is obtained. Thecarboxymethylchitosan so formed will be called "partially N-acetylatedcarboxymethylchitosan".

The partially N-acetylated carboxymethylchitosan is next dissolved, forexample, in a mixed solvent of dimethylformamide and 0.5% aq. NaHCO₃solution, followed by adding thereto an N-hydroxysuccinimido ester ofsuch a peptide which is usable in this invention and which peptide hashad a protective group introduced into the amino group of the terminalamino acid of the peptide chain and will hereinafter be called simplythe "protected peptide" that can be represented by a formula: "HO--X--protective group". Said N-hydroxysuccinimido ester is an active ester ofthe protected peptide. They are reacted with each other so that the"protected peptide" can be linked to the free amino groups of thepartially N-acetylated carboxymethylchitosan. By changing the amount ofthe active ester of the "protected peptide" to be added, a complex of"partially-N-acetylated-carboxymethylchitosan-protected peptide" havingthe protected peptide linked in varying amounts can be formed. If theactive ester of the protected peptide is reacted in a large excess, itis possible to obtain a complex where the free amino groups do not existpractically. The active ester of the protected peptide can be obtainedin a manner known per se in the field of peptide synthesis, for example,by dissolving the protected peptide in dimethylformamide, addingN-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide to the solutionand then reacting them.

The concentration of the partially N-acetylated carboxymethylchitosanwhich is employed in the reaction mixture for the above reaction linkingto the active ester of the protected peptide as described above maysuitably be 0.1-5% (wt. %). The molar ratio of the sugar residues of thepolysaccharide (the chitosan) to the active ester of the protectedpeptide may appropriately range from 20:1 to 1:10. The number of aminoacids present in the molecule of the protected peptide is in a range of1-10.

When the above-mentioned linking reaction is made by using apharmaceutical compound, namely a drug compound in place of theprotective groups, that is to say, by adding the N-hydroxysuccinimidoester of such a peptide which is usable in this invention and whichpeptide has the drug compound introduced therein and is represented by aformula: "HO--X--P" where P denotes the residue R₃ CO--, the residue R₄NH-- or the residue R₅ O-- originated from the pharmaceutical or drugcompound, there is formed a complex of"partially-N-acetylated-carboxymethylchitosan-peptide-drug".

Further, subsequent removal of the protective groups or the drug residue(--P) from the complex which has been formed as above will afford acomplex of "partially-N-acetylated-carboxymethylchitosan-peptide".

The compound of the formula (II) provided according to the presentinvention may embrace three types of the substances as formed and asdescribed above, and are thus the"partially-N-acetylated-carboxymethylchitosan-protected peptide"complex, the "partially-N-acetylated-carboxymethylchitosan-peptide-drug"complex, and the "partially-N-acetylated-carboxymethylchitosan-peptide"complex.

The compound of the formula (II) according to this invention can beconverted to the corresponding compound of the formula (I) according tothis invention when the former compound (II) is dissolved in a saturatedaqueous solution of NaHCO₃ and is then reacted with acetic anhydride oranother suitable acetylating agent, for example, acetyl chloride toacetylate the free amino groups which are remaining in the complexcompound of the formula (II). However, the compound obtained here issuch a compound of the formula (I) where either a=a₁ and a₂ =0, or a=a₂and a₁ =0. Corresponding to the above-described three types of thecompound of the formula (II), for example, a complex of"N-acetyl-carboxymethylchitosan-protected peptide", a complex of"N-acetylcarboxymethylchitosan-peptide-drug" and a complex of"N-acetylcarboxymethylchitosan-peptide" can be produced through theacetylation, respectively. The former two complexes just mentioned aboveare the corresponding compound of the formula (I) where a=a₁ and a₂ =0,whereas the last-mentioned complex is the corresponding compound of theformula (I) where a=a₂ and a₁ = 0. Further, the former two complexes canbe converted into the last-mentioned complex generally by acidtreatment. For example, the "N-acetylcarboxymethylchitosan-peptide"complex can be produced by subjecting the corresponding"N-acetylcarboxymethylchitosan-protected peptide" complex to mild acidtreatment, for example, by treating the latter complex in 0.5N HCl at30° C. for 16 hours.

The "N-acetylcarboxymethylchitosan-peptide-drug" complex can be producedby subsequently dissolving the above described"N-acetylcarboxymethylchitosan-peptide" complex, for example, in 1%aqueous NaHCO₃ solution and then reacting the N-terminal of the peptidechain of the latter complex with a drug compound having the carboxylgroup in the form of the N-hydroxysuccinimido ester (active ester) ofthe drug compound having the carboxyl group. It is however to be notedthat the compound (I) as produced here is not necessarily limited to thecompound of the formula (I) where a=a₁ and a₂ =0 but, in practice, maybe indicated as such compound of the formula (I) where a=a₁ +a₂ and a₂≠0. Thus, for instance, if methotrexate (MTX) is chosen as such drugcompound here and reacted with the peptide chain of the complex, the"N-acetylcarboxymethylchitosan-peptide--MTX" complex can be produced.The content of MTX so introduced in the latter complex produced mayusually be lower than the content of the peptide chain X in the complex.

In a third aspect of the present invention, therefore, there is provideda process for the preparation of a partially N-acetylatedcarboxymethylchitosan derivative represented by the following formula(II): ##STR4## wherein R₁ and R₂ individually mean H or carboxymethylgroup; P denotes H, a group OH, a group R₃ CO--, a group R₄ NH-- or agroup R₅ O-- with assuming that R₃ COOH denotes a compound havingcarboxyl group, R₄ NH₂ denotes a compound having amino group and R₅ OHdenotes an alcohol compound; X represents a peptide chain containingsame or different, 1-10 amino acids; and a, b₁ and b₂ individuallyrepresent a positive integer; and having the following characteristicvalues (1)-(4):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a/(a + b):           0.01-1                                                   [provided that b = b.sub.1 + b.sub.2 ]                                 (4)    P/X ratio (molar ratio):                                                                           0.1-1                                             ______________________________________                                    

characterized in that the process comprises reacting a compound havingthe following formula:

    PX--OH                                                     (IV)

wherein P and X have the same meanings as defined above with a partiallyN-acetylated carboxymethylchitosan having the following formula (III):##STR5## wherein R₁ and R₂ have the same meanings as defined above andhaving the following characteristic values (1)-(2):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000.                                           by gel filtration method):                                             ______________________________________                                    

In a fourth aspect of the present invention, there is also provided aprocess for the preparation of an N-acetylcarboxymethylchitosanderivative represented by the following formula (I): ##STR6## wherein R₁and R₂ individually mean H or carboxymethyl group; P denotes a group R₃CO--, a group R₄ NH-- or group R₅ O-- with assuming that R₃ COOH denotesa compound having carboxyl group, R₄ NH₂ denotes a compound having aminogroup and R₅ OH denotes an alcohol compound; Q stands for H or a group--OH; X represents a peptide chain containing same or different, one toten amino acids; a₁ and a₂ individually represent zero or a positiveinteger, provided that both of a₁ and a₂ are not zero at the same time,and b stands for a positive integer; and having the followingcharacteristic values (1)-(4):

    ______________________________________                                        (1)   carboxymethylation degree:                                                                       0.5-1.2                                              (2)   molecular weight (as measured                                                                     3,000-300,000                                             by gel filtration method):                                              (3)   a/(a + b):         0.01-1                                                     [provided that a = a.sub.1 + a.sub.2 ]                                  (4)   P/X ratio (molar ratio):                                                                         0.1-1                                                ______________________________________                                    

characterized in that the process comprises:

acetylating a partially N-acetylated carboxymethylchitosan derivativehaving the following formula (II') ##STR7## wherein R₁ and R₂individually mean H or carboxymethyl group; P₁ denotes H, a group OH, agroup R₃ 'CO--, a group R₄ 'NH-- or a group R₅ 'O-- with assuming thatR₃ 'COOH denotes a compound having carboxyl group, R₄ 'NH₂ denotes acompound having amino group and R₅ 'OH denotes an alcohol compound; Xrepresents a peptide chain containing same or different, one to tenamino acids; and a, b₁ and b₂ individually represent a positive integer;and having the following characteristic values (1)-(4):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a/(a + b):           0.01-1                                                   [provided that b = b.sub.1 + b.sub.2 ]                                 (4)    P.sub.1 /X ratio (molar ratio):                                                                    0.1-1,                                            ______________________________________                                    

removing the P₁ groups from the resulting acetylation product, where theP₁ groups are other than H or the group --OH; and

reacting the resulting reaction product having the P₁ groups removed,with a compound of the following formula:

    P--H or P--OH                                              (V)

wherein P means the group R₃ CO--, the group R₄ NH-- or the group R₅ O--as defined above.

We, the present inventors, have also proceeded with research to furtherimprove the properties of the N-acetylcarboxymethylchitosan derivativeof formula (I) according to the first aspect of this invention. As aresult, we have succeeded in synthesizing as a novel substance such anN-acetylcarboxymethylchitosan derivative represented by the followingformula (X): ##STR8## wherein R₁, R₂, P, Q and X have the same meaningsas defined above in the formula (I) , PEG represents a group having thefollowing formula (VI) , (VII) , (VIII) or (IX): ##STR9##

    --CO--CH.sub.2 CH.sub.2 --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (VII)

    --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3            (VIII)

    --CO--CH.sub.2 --O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (IX)

in which n means the average polymerization degree of the polyethyleneglycol chain; a₁ and a₂ individually represent 0 or a positive integerprovided that both of a₁ and a₂ are not 0 at the same time; b and cindividually stand for a positive integer; and having the followingcharacteristic values (1)-(4):

    ______________________________________                                        (1)   carboxymethylation degree:                                                                       0.5-1.2                                              (2)   molecular weight (as measured                                                                     3,000-300,000                                             by gel filtration method):                                              (3)   a/(a + b + c):     0.01-1                                                     [provided that a = a.sub.1 + a.sub.2 ]                                  (4)   P/X ratio (molar ratio):                                                                         0.1-1                                                ______________________________________                                    

by replacing a plurality of the N-acetylcarboxymethylglucosamine units,namely the sugar units contained in the drivative of the formula (I), bya corresponding plurality of such carboxymethylglucosamine units havingtheir 2-amino groups substituted with a group which possesses apolyethylene glycol chain therein (hereinafter, said group may beabbreviated simply as "PEG"), specifically by such apolyethlene-glycol-substituted carboxymethylglucosamine units having theformula (A): ##STR10## wherein R₁, R₂, PEG and n have the same meaningsas defined above. Moreover, it has been found that the novelN-acetylcarboxymethylchitosan derivative represented by the formula (X)can exhibit a solubility in water as enhanced much than theN-acetylcarboxymethylchitosan derivative of the formula (I) itself, andis usable as a carrier for the delivery of a drug compound and also canreside or remain for a long time in blood after its administration.

We, the present inventors, have also succeeded in synthesizing as anovel substance such an N-acetylcarboxymethylchitosan derivativerepresented by the following formula (XI): ##STR11## wherein R₁, R₂, P,Q and X have the same meanings as defined above in the formula (I); a₁and a₂ individually represent 0 or a positive integer provided that bothof a₁ and a₂ are not 0 at the same time; and b and c individually standfor a positive integer; and having the following characteristic values(1)-(4):

    ______________________________________                                        (1)   carboxymethylation degree:                                                                       0.5-1.2                                              (2)   molecular weight (as measured                                                                     3,000-300,000                                             by gel filtration method):                                              (3)   a/(a + b + c):     0.01-1                                                     [provided that a = a.sub.1 + a.sub.2 ]                                  (4)   P/X ratio (molar ratio):                                                                         0.1-1                                                ______________________________________                                    

by replacing a plurality of the N-acetylcarboxymethylglucosamine units,namely the constituent sugar units of the derivative of the formula (I),by a corresponding plurality of polyol units having the formula (B):##STR12## wherein R₁ has the same meanings as defined above. Moreover,it has been found that the novel N-acetylcarboxymethylchitosanderivative represented by the formula (XI) can exhibit a solubility inwater as enhanced much than the N-acetylcarboxymethylchitosan derivativeof the formula (I) itself, and is usable as a carrier for the deliveryof a drug compound and can remain and retain for a long time in bloodafter its administration of the derivative of formula (XI).

In a fifth aspect of the present invention, therefore,there is providedan N-acetylcarboxymethylchitosan derivative represented by the followingformula (X) or (XI): ##STR13## wherein R₁ and R₂ individually mean H orcarboxymethyl group; P denotes a group R₃ CO--, a group R₄ NH-- or agroup R₅ O-- with assuming that R₃ COOH denotes a compound havingcarboxyl group, R₄ NH₂ denotes a compound having amino group and R₅ OHdenotes an alcohol compound; Q stands for H or a group --OH; Xrepresents a peptide chain containing same or different, one to tenamino acids; a₁ and a₂ individually represent zero or a positiveinteger, provided that both of a₁ and a₂ are not zero at the same time;b and c individually stand for a positive integer, and -PEG represents agroup having the following formula (VI), (VII), (VIII) or (IX):##STR14##

    --CO--CH.sub.2 CH.sub.2 --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3(VII)

    --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3            (VIII)

    --CO--CH.sub.2 --O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (IX)

in which n means the average polymerization degree of the polyethyleneglycol chain; and having the following characteristic values (1)-(4):

    ______________________________________                                        (1)   carboxymethylation degree:                                                                       0.5-1.2                                              (2)   molecular weight (as measured                                                                     3,000-300,000                                             by gel filtration method):                                              (3)   a/(a + b):         0.01-1                                                     [provided that a = a.sub.1 + a.sub.2 ]                                  (4)   P/X ratio (molar ratio):                                                                         0.1-1                                                ______________________________________                                    

The derivative of the formula (X) according the fifth aspect of thisinvention is such an N-acetylcarboxymethylchitosan derivative of theformula (I) where some of the sugar units have been replaced by sugarunits of the formula (A) given below. On the other hand, the derivativeof the formula (XI) according to the fifth aspect of this invention issuch an N-acetylcarboxymethylchitosan derivative where some of the sugarunits have been replaced likewise by polyol units of the formula (B)given below. Examples 17-21 and Examples 22-23, which will be describedhereinafter, demonstrate some examples of the compound of the formula(X) and some examples of the compound of the formula (XI), respectively.##STR15##

The sugar derivative of the above formula (A) is such a sugar unit inwhich the 2-amino group of the carboxymethylglucosamine unit has beensubstituted by PEG. Specifically, PEG represents one of groups havingthe following formulae (VI) to (IX) and containing a polyethylenegloycol chain: ##STR16##

    --CO--CH.sub.2 CH.sub.2 --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3(VII)

    --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3            (VIII)

    --CO--CH.sub.2 --O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (IX)

The polyethlene glycol chain contained in each of the groups of theformulae (VI) to (IX) may have various molecular weights, but thepolyethylene glycol chain which is preferred from the standpoint of itsgeneral availability is such that having an average molecular weight ofabout 5,000. Accordingly, the preferred average polymerization degree nof the polyethylene glycol chain present in each group of the formulae(VI)-(IX) is about 100-120, to which this invention is not limited,however. The content of the polyethylene glycol in the derivative of theformula (X) can be determined by an NMR method from measurement of thearea of a peak attributable to methylene protons in the polyethyleneglycol chain. Greater contents of the polyethylene glycol are preferredand for example, its contents of 1% (by weight) or higher aresatisfactory. However, no particular limitation is imposed on thecontent of polyethylene glycol in this invention.

On the other hand, the polyol unit of the formula (B) in the derivativeof the formula (XI) according to the fifth aspect of this invention hasa chemical structural feature in that the bond between the carbon atomsat the 2-and 3-positions of the carboxymethylglucosamine unit has beencleaved by oxidation and, subsequent to the cleavage, thecarboxymethylglucosamine unit has been converted into the form of apolyalcohol by reduction.

The a/(a+b+c) value in each of the derivatives of the formulae (X) and(XI) according to the fifth aspect of this invention shows thepeptide-introducing degree which may be defined similarly to the a/(a+b)value for the formula (I). The a/(a+b+c) value can therefore bedetermined using the above equation (1) as it is. In practice, thisvalue may be calculated in exactly the same manner as for the compoundof the formula (I), while using the above equations (2)-(5). Similarly,the ranges of the carboxylmethylation degree and molecular weight (asmeasured by gel filtration method) of the derivatives of formulae (X)and (XI) can also be same as those of the compound of the formula (I).

Production of the compound of the formula (X) according to the fifthaspect of this invention is conducted by preparing as a startingmaterial the "partially-N-acetylated-carboxymethylchitosan-protectedpeptide" complex of the formula (II) and then treating it, as will bedescribed below. Thus, first, an active derivative of a PEG chain isreacted with the non-N-acetylated and free 2-amino-group-containingcarboxymethylglucosamine units existing in the complex of the formula(II) so as to introduce the PEG chain of anyone of the formula (VI)-(IX)as a substituent group into some of the free 2-amino groups of saidunits. Further, any remaining free amino groups of thecarboxymethylgluocosamine units of said complex are N-acetylated. Theprotective groups are then removed from the moiety of the protectedpeptide present in the N-acetylated complex to achieve the deprotectionof the latter. A reactive derivative of a drug compound is then reactedwith the free terminal of the deprotected peptide moiety of the complexso that the drug compound (P) is linked to the terminal of the peptidemoiety of the complex.

In the above reaction steps, the various intermediates as formed afterthe above N-acetylation and the final product as linked to the drugcompound may generically be represented by the formula (X) shown in theabove.

The active derivative of the PEG chain can be, for example, an activeester (for example, the N-hydroxysuccinimido ester) of2-O-mono-methyoxy-polyethylene glycol-3, 5-dichloro-s-triazine ormethoxy-polyoxyethylene carboxylic acid.

For the production of the compound of the formula (XI) according to thefifth aspect of this invention, the"partially-N-acetylated-carboxymethylchitosan-protected peptide" complexof the formula (II) is prepared at first as a starting materialsimilarly to the production of the compound of the formula (X). Thestarting material is then treated, as will be described next. Thus,periodic acid, for example, is first reacted with the non-N-acetylatedand free amino-group-containing carboxymethylglucosamine units of thestarting complex of formula (II) so that the pyranose rings of theglucosamine are cleaved by oxidation. The aldehyde groups as formed bythe cleavage is then reduced, for example, with sodium borohydride sothat the complex of formula (II) is converted into the form of a polyol.The protective groups are then removed from the protected peptide moietyof the complex to deprotect the peptide moiety of the complex. Areactive derivative of a drug compound is then reacted with the freeterminals of the deprotected peptide moiety, whereby the drug compound(P) is linked to the terminals of the peptide moieties of the complex.In the above reaction steps, the various intermediate as formed afterthe conversion of the complex (II) into the polyol and the final productas linked to the drug compound may generically be represented by theformula (XI) above.

The substances of formulae (X) and (XI) according to the fifth aspect ofthe present invention have a high solubility in water, and thereforesuch drug-delivering carriers and their complexes linked to a drug,which have improved water solubility, are provided by the compound offormula (X) or (XI) according to the fifth aspect of this invention.According to Experiment 4 to be described hereinafter, it is revealedthat the substance of formula (X), for example, is excellent in itsproperty of residing or remaining in blood and exhibits an improvedselectivity to be delivered toward the site of cancer (theorganotropism) when said substance bears an anticancer agent as a drugcompound or pharmaceutical compound.

The derivative of the formula (I) and the derivatives of the formulae(X) and (XI), all of which pertain to this invention, have advantageousproperties, as will be described below.

As will be demonstrated by the experiments to be described hereinafter,the derivatives of the formula (I) and (X) according to this inventioncan remain stable in blood during the period after their administrationby intravenous injection until their arrival at a target organ, and inother words, said derivatives can retain their high level required ofthe substances of this invention in blood. On the other hand, they canundergo gradual enzymatic degradation in vivo, so that there is noproblem that the N-acetylcarboxymethylchitosan unit of this derivativewould remain objectionably for a long time in the living body. Further,all the derivative of the formula (I) and the derivatives of theformulas (X) and (XI) according to this invention, can be observed topossess the tendency of organotropism.

Incidentally, that the tendency of organotropism is possessed by thecompounds of the formulae (I), (X) and (XI) according to this inventionwhen they are administered, does neither mean anything beyond that saidcompounds can exhibit a tendency of increasing the concentrations of thepresent compounds as measured at the site of a particular target organ,in comparison with such cases when there are administered such similarcompounds which are prepared not in accordance with this invention, norit does mean that the present compounds of this invention canselectively be delivered only to the site of the particular targetorgan.

The present invention will hereinafter be described more specifically bythe following Examples.

The first to fourth aspects of the present invention are illustrated byExamples 1-16, while the fifth aspect of the present invention isillustrated by Examples 17-23.

In each Example below, the gel filtration was conducted under thefollowing conditions: a column of TSK-gel G4000PW_(XL) was used with aneluent of 0.1M NaCl at a flow rate of 0.8 ml/min. and a columntemperature of 40° C., and with an amount of the sample injected beingabout 75 μg.

EXAMPLE 1

5.0 Grams of carboxymethylchitin (its carboxymethylation degree was 1.0per one sugar reside, commercially available from FunakoshiPharmaceutical Co., Ltd.) were dissolved in 0.05M acetate buffer (havinga pH 6.0, 500 ml), to which egg-white lysozyme (12.5 mg) was then added,followed by incubation at 37° C. for 2 hours for the reaction. Thereaction solution obtained was added to ethanol (2 l). The resultingprecipitate was collected and dried in vacuo, whereby 4.25 g of adepolymerised carboxymethylchitin were obtained. Thiscarboxymethylchitin (3.9 g) was dissolved in 1N aq. NaOH solution (390ml) and the resultant mixture was then heated under reflux at 100° C.for 6 hours. After the pH of the resulting reaction solution wasadjusted to 8, the reaction solution was centrifuged to give asupernatant. The supernatant was added to methanol (1.9 l) and theresulting precipitate was collected. The precipitate was dried in vacuoto afford 1.91 g of partially N-acetylated carboxymethylchitosan, whichmay hereinafter be called simply as a "polysaccharide" and which is oneexample of the substances of the formula (III). The molecular weight ofthis polysaccharide was found to be about 1×10⁵, as measured by gelfiltration method (column: G4000PW_(XL)) while using a dextran asstandard substance.

The above polysaccharide (200 mg) was dissolved in 0.5% aq. NaHCO₃solution (20 ml), followed by addition of dimethylformamide (17.5 ml)into the resultant uniform solution of the polysaccharide. On the otherhand, a peptide of which the terminal amino group has been protected bytert-butoxycarbonyl group (namely, Boc), and which was shown by formulaN-Boc-Phe-Phe-Gly-OH, (94 mg), was dissolved in 1.5 ml ofdimethylformamide, followed by addition of N-hydroxysuccinimide (23 mg)and N,N'-dicyclohexylcarbodiimide (37 mg) to the resulting solution. Theprotected peptide and the chemical reagents added were reacted with eachother at 4° C. for 24 hours to form an active ester of the N-protectedpeptide. The whole reaction mixture was added to the above-mentionedpolysaccharide solution, followed by reaction at 4° C. for 16 hours. Theresulting reaction solution was added to ethanol (160 ml). The resultingprecipitate was collected and then dried in vacuo, thereby to afford 200mg of a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc [as oneexample of the derivative of the formula (II)].

The above complex (150 mg) was dissolved in saturated aq. NaHCO₃solution (15 ml), to which acetic anhydride (0.6 ml) was then added,followed by the N-acetylation of the complex being conducted at roomtemperature for 17 hours. The resulting reaction solution wasneutralized and then poured into ethanol (80 ml). The resultingprecipitate was collected and then dried in vacuo, thereby to afford 154mg of a complex of N-acetylcarboxymethylchitosan--Gly-Phe-Phe-Boc as oneexample of the derivatives of the formula (I). Ultraviolet absorptionspectrum and GFC (gel filtration chromatographic) elution pattern of theabove complex as obtained are shown in FIG. 1 and FIG. 2 of theaccompanying drawings, respectively. The content of the N-Boc-peptide inthis complex was determined to be 14.1% (wt. %), by ultravioletabsorption spectrophotometry (at 258 nm).

The above complex (130 mg) was dissolved in 0.5N HCl (13 ml) and thenreacted at 30° C. for 17 hours, so that the complex was subjected to thedeprotecting treatment for removal of the Boc groups from the complex.The reaction solution obtained was neutralized and then poured intoethanol (70 ml). The resulting precipitate was collected and then driedin vacuo, thereby to afford 121 mg of a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Phe-H [as another example of thederivative of the formula (I)]. Ultraviolet absorption spectrum and GFCelution pattern of this complex as afforded are shown in FIG. 3 and FIG.4 of the accompanying drawings, respectively. The content of the peptidechain in this complex was determined to be 11.3% (wt. %), by ultravioletabsorption spectrophotometry (258 nm).

Methotrexate (MTX) (182 mg) as a pharmaceutical or drug compound havingthe carboxyl group was dissolved in dimethylformamide (4 ml), to whichN,N'-dicyclohexylcarbodiimide (82 mg) was then added, followed byreaction at 4° C. for 17 hours. N-Hydroxysuccinimide (46 mg) andpyridine (63 μl) were added to the resulting reaction solution, and thereaction mixture so formed was then subjected to the reaction at roomtemperature for 5 hours so that an active ester of MTX was prepared inthe resulting reaction solution. On the other hand, the above complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Phe-H, (50 mg) was dissolved in0.5% aq. NaHCO₃ solution (10 ml), followed by addition thereto of 0.5 mlof the above-mentioned reaction solution which contained the activeester of MTX. They were reacted with each other at 4° C. for 15 hours,whereby MTX was linked to N-terminals of the peptide chain-Gly-Phe-Phe-H of the complex. Then, the reaction solution so obtainedwas poured into ethanol (40 ml). The resulting precipitate was collectedand then dried in vacuo, to obtain 53 mg of a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Phe--MTX [as a further example ofthe complex of the formula (I)] as yellow powder. Ultraviolet-visibleabsorption spectrum and GFC elution pattern of this complex are shown inFIG. 5 and FIG. 6 of the accompanying drawings, respectively. Thecontent of MTX in this complex was determined to be 11.5% (wt. %), byultraviolet absorption spectrophotometry (at 307 nm). Further, the P/Xmolar ratio of this complex, more specifically, the ratio of MTX/peptide(molar ratio) in this complex was 0.93 as calculated in accordance withthe calculation equation (5) described hereinbefore.

With respect to this complex, the value of a/(a+b) as defined for theformula (I) was approximately 0.09 as calculated in accordance with thecalculation equation (1) given before.

EXAMPLE 2

The partially N-acetylated carboxymethylchitosan (100 mg) obtained inExample 1 above was dissolved in 1% aq. NaHCO₃ solution (10 ml),followed by addition of dimethylformamide (6 ml) into the resultinguniform polysaccharide solution. A peptide of which terminal amino groupwas protected by p-methoxybenzyloxycarbonyl group (pMZ), and which wasshown by a formula pMZ-Gly-Gly-Gly-OH, (141 mg), was dissolved in 4 mlof dimethylformamide, to which N-hydroxysuccinimide (46 mg) andN,N'-dicyclohexylcarbodiimide (74 mg) were then added. They were reactedat room temperature for 3 hours to form an active ester of theN-protected peptide. The whole reaction solution containing the aboveactive ester was added to the above-mentioned polysaccharide solution,to which dimethylformamide (10 ml) was then added. The whole mixture wassubjected to reaction at 4° C. for 23 hours. Water (5 ml) was added tothe resulting reaction solution, followed by centrifugation to obtain asupernatant. The supernatant was added to ethanol (150 ml) and theresulting precipitate was collected. The precipitate was then dried invacuo, thereby to afford 120 mg of a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Gly-pMZ [as oneexample of the derivatives of the formula (II)].

In a similar manner to Example 1, this complex (100 mg) was N-acetylatedwith acetic anhydride, whereby a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-pMZ, (97 mg) was obtained.Ultraviolet absorption spectrum and GFC elution pattern of the resultingcomplex as acetylated are shown in FIG. 7 and FIG. 8, respectively. Thecontent of the pMZ-peptide in this complex was determined to be 20.1%(wt. %) by ultraviolet absorption spectrophotometry (at 272 nm).

In a similar manner to Example 1, this complex (89 mg) was subjected toacid treatment to remove pMZ, the protective groups, whereby 78 mg of acomplex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-H were obtained.Ultraviolet absorption spectrum of this complex are shown in FIG. 9. Thecontent of the peptide chain in this complex was 11.3% (wt. %) asdetermined in accordance with the calculation equation (3) givenhereinbefore.

50 mg of this complex were dissolved in 1% aq. NaHCO₃ solution (5 ml),to which a solution (1 ml) of the active ester of MTX was then added,followed by the reaction at 4° C. for 21 hours. The resulting reactionsolution was poured into ethanol (25 ml). The resulting precipitate wascollected and then dried in vacuo, to afford 52 mg of a complex ofN-acetylcarboxymethylchiotosan--Gly-Gly-Gly--MTX [as a further exampleof the complex of the formula (I)] as yellow powder. Ultraviolet-visibleabsorption spectrum and GFC elution pattern of this complex are shown inFIG. 10 and FIG. 11, respectively. The content of MTX in this complexwas determined to be 21.4% (wt. %) by ultraviolet absorptionspectrophotometry (at 307 nm). Further, the P/X molar ratio of thiscomplex, more specifically, the ratio of MTX/peptide (molar ratio) insaid complex was 1.0 as calculated in accordance with the calculationequation (5) described hereinbefore. With respect to said complex, itsvalue of a/(a+b) as defined for the formula (I) was 0.2 as calculated inaccordance with the calculation equation (1) given hereinbefore.

EXAMPLE 3

Egg white lysozyme was reacted with 5.0 g of a carboxymethylchitin (itscarboxymethylation degree was 1.0 per one sugar residue) in a similarmanner to Example 1 to prepare a depolymerised carboxymethylchitin (4.28g). A 4.1 g portion of this depolymerised carboxymethylchitin wassubjected to alkali treatment, whereby a partially N-acetylatedcarboxymethylchitosan (2.22 g) having a molecular weight of about 1×10⁵was obtained.

An active ester of an N-protected peptide of formulaN-Boc-Gly-Phe-Gly-Gly-OH (90 mg) was reacted with the partiallyN-acetylated carboxymethylchitosan (200 mg) as obtained above in asimilar manner to Example 1 to produce a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Phe-Gly-Boc, (210mg). A 200 mg portion of this complex was thereafter N-acetylated toprepare a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly-Boc,(190 mg). The content of the N-Boc-peptide chain in this complex wasdetermined to be 9.8 wt. % (wt. %) by ultraviolet absorptionspectrophotometry (at 258 nm).

A 160 mg portion of this complex was deprotected by acid treatment in asimilar manner to Example 1 to obtain a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly-H, (144 mg, with apeptide content of 7.6%).

An active ester of MTX (55 mg) was reacted with a 120 mg portion of thelatter complex, whereby a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly--MTX (127 mg, with an MTXcontent of 9.5%) was produced. With respect to this complex, the ratioof MTX/peptide (molar ratio) in said complex and its value of a/(a+b)defined for the formula (I) were 1.0 and 0.07, respectively, ascalculated in accordance with the aforesaid calculation equations (5)and (1).

EXAMPLE 4

In a similar manner to Example 1, an active ester of an N-protectedpeptide,N-Boc-Gly-Phe-Gly-Phe-OH, (105 mg) was reacted with thepartially N-acetylated carboxymethylchitosan (200 mg) which was obtainedin Example 3 above. Thereby, a complex ofpartially-N-acetylated-carboxymethylchitosan--Phe-Gly-Phe-Gly-Boc, (199mg) was produced.

A 188 mg portion of this complex was N-acetylated so that a complex ofN-acetylcarboxymethylchitosan--Phe-Gly-Phe-Gly-Boc, (191 mg) wasprepared. The content of the N-Boc-peptide chain in this complex wasdetermined to be 4.9% (wt. %) by ultraviolet absorptionspectrophotometry (at 258 nm). A 160 mg portion of this complex wasdeprotected by acid treatment in a similar manner to Example 1 to afforda complex of N-acetylcarboxymethylchitosan--Phe-Gly-Phe-Gly-H, (150 mg,with a peptide content of 4.0%). An active ester of MTX (23 mg) wasthereafter reacted with a 50 mg portion of the latter complex, whereby acomplex of N-acetylcarboxymethylchitosan--Phe-Gly-Phe-Gly--MTX (46 mg,with an MTX content of 4.5 wt. %) was prepared. With respect to thiscomplex, the MTX/peptide molar ratio in this complex and its value ofa/(a+b) defined for the formula (I) were 1.1 and 0.03, respectively, ascalculated from the aforesaid calculation equations (5) and (1).

EXAMPLE 5

In a similar manner to Example 1, an active ester of an N-protectedpeptide, N-Boc-Phe-Gly-Phe-Gly-OH, (54 mg) was reacted with thepartially N-acetylated carboxymethylchitosan (100 mg) as obtained inExample 3. Thus, a complex ofpartially-N-acetylated-carboxymethylchitosan-Gly-Phe-Gly-Phe-Boc, (109mg) was prepared.

A 90 mg portion of this complex was N-acetylated, affording a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe-Boc, (85 mg). The contentof the N-Boc-peptide chain in this complex was determined to be 12.7%(wt. %) by ultraviolet absorption spectrophotometry (at 258 nm). A 72 mgportion of this complex was deprotected by acid treatment in a similarmanner to Example 1 to prepare a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe-H, (63 mg, with a peptidecontent of 10.4%).

An active ester of MTX (23 mg) was thereafter reacted with a 50 mgportion of the latter complex, whereby a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe--MTX (49 mg, with an MTXcontent of 9.4%) was produced. With respect to this complex, the ratioof MTX/peptide (molar ratio) in said complex and its value a/(a+b)defined for the formula (I) were 0.94 and 0.07, respectively, ascalculated in accordance with the aforesaid equations (5) and (1).

EXAMPLE 6

In a similar manner to Example 1, an active ester of an N-protectedpeptide, N-Boc-Ala-Gly-Gly-Gly-OH, (180 mg) was reacted with thepartially N-acetylated carboxymethylchitosan (200 mg) as obtained inExample 3, whereby a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Gly-Ala-Boc, (218mg) was prepared. A 150 mg portion of this complex was N-acetylated,affording a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-Boc, (143 mg). A 130 mg portion of the lattercomplex was subjected to acid treatment for the deprotection in asimilar manner to Example 1 to produce a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-H, (120 mg).

The active ester of MTX (46 mg) was thereafter reacted with a 50 mgportion of the last-mentioned complex, whereby a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala--MTX (55 mg, with an MTXcontent of 21.5%) was obtained. Ultraviolet-visible absorption spectrumand GFC elution pattern of the latter complex are shown in FIG. 12 andFIG. 13 of the accompanying drawings, respectively.

EXAMPLE 7

Partially N-acetylated carboxymethylchitosan (45 mg), which had beenprepared from carboxymethylchitin (its carboxymethylation degree was 0.7per sugar residue) in a similar manner to Example 1 and had a molecularweight of about 1×10⁵ was dissolved in a 0 1M borate buffered solution(pH 8.0, 2 ml) to prepare a solution of the polysaccharide. On the otherhand, a modified peptide of a formulaN-succinyl-Ala-Ala-Ala-p-nitroanilide, (45 mg) was dissolved indimethylformamide (2 ml), to which were then added N-hydroxysuccinimide(12 mg) and N,N'-dicyclohexylcarbodiimide (41 mg). The reaction was madeat room temperature for 1 hour and then at 4° C. for 18 hours, so thatan active ester of said peptide having the nitroanilide bonded theretowas prepared. The resulting reaction solution was distilled underreduced pressure to remove the solvent therefrom. The residue was washedwith isopropanol and then dissolved in dimethylformamide (0.8 ml). Thesolution obtained was added to the above-mentioned polysaccharidesolution, followed by reaction at room temperature for 40 hours. Ethanolwas added to the resulting reaction solution. The precipitate as formedwas collected and then dried in vacuo, whereby 46 mg of a complex ofpartially-N-acetylated-carboxymethylchitosan--Suc-Ala-Ala-Ala--p-nitroanilidewere obtained.

26 mg of this complex was taken and dissolved in saturated aq. NaHCO₃solution (3 ml), to which acetic anhydride (0.14 ml) was then added,followed by the reaction at 4° C. for 24 hours to N-acetylate thecomplex. After the reaction solution obtained was dialyzed againstwater, the resulting non-dialysable solution left after dialysis waspoured into ethanol. The resulting precipitate was dried in vacuo sothat a complex ofN-acetylcarboxymethylchitosan--Suc-Ala-Ala-Ala--p-nitroanilide (25 mg)was obtained. Ultraviolet absorption spectrum and GFC elution pattern ofthis complex are shown in FIG. 14 and FIG. 15 of the accompanyingdrawings, respectively. The content of the moietySuc-Ala-Ala-Ala--p-nitroanilide in this complex was found to be 3.9%(wt. %) from its absorption spectrophotometry at 315 nm.

EXAMPLE 8

An active ester of a modified peptide of formulaN-succinyl-Ala-Ala-Val-Ala--p-nitroanilide, (55 mg) was reacted with thepartially N-acetylated carboxymethylchitosan (45 mg) in a similar mannerto Example 7 to prepare a complex ofpartially-N-acetylated-carboxymethylchitosan--Suc-Ala-Ala-Val-Ala--p-nitroanilide(44 mg).

30 mg of this complex was thereafter N-acetylated in a similar manner toExample 7, to give a complex ofN-acetylcarboxymethylchitosan--Suc-Ala-Ala-Val-Ala--p-nitroanilide (22mg). The content of the moiety Suc-Ala-Ala-Val-Ala--p-nitroanilide inthe latter complex was found to be 4.5% (wt. %) from its absorptionspectrophotometry at 315 nm.

EXAMPLE 9

The partially N-acetylated carboxymethylchitosan (50 mg) as obtained inExample 1 was dissolved in 0.25% aq. NaHCO₃ solution (7 ml), and intothe resulting uniform solution of the polysaccharide was addeddimethylformamide (6 ml). On the other hand, a phenylalanine having beenprotected by a protective group Boc and shown by a formula N-Boc-Phe-OH(256 mg) was dissolved in 3 ml of dimethylformamide, followed byaddition of N-hydroxysuccinimide (115 mg) andN,N'-dicyclohexylcarbodiimide (190 mg) to the resultant solution. Thereaction was made at 4° C. for 20 hours to prepare an active ester ofthe N-protected phenylalanine. A 1 ml portion of the resultant reactionsolution which contained said active ester was added to theabove-mentioned polysaccharide solution, followed by the reaction atroom temperature for 20 hours. The reaction solution obtained was addedto ethanol (35 ml). The resulting precipitate was collected and thendried in vacuo, whereby 51 mg of a complex ofpartially-N-acetylated-carboxymethylchitosan--Phe-Boc were obtained. Thecontent of the N-Boc-phenylalanine (Phe-Boc) in this complex wasdetermined to be 14.4% (wt. %) from ultraviolet absorptionspectrophotometry (at 258 nm).

EXAMPLE 10

The depolymerized carboxymethylchitin (4.1 g) as prepared in a similarmanner to Example 3 was partially de-N-acetylated by alkali treatmentand the resulting reaction solution was adjusted to pH 8.5. The reactionsolution was then centrifuged and the supernatant was added to 4.4-foldvolumes of methanol. The resulting mixture was separated into thesupernatant and precipitate. Ethanol (300 ml) was added to thesupernatant. The resulting precipitate was collected and then dried invacuo so that 0.54 g of partially-N-acetylated-carboxymethylchitosanhaving a molecular weight of about 2×10⁴ was obtained.

This chitosan substance (100 mg) was reacted with an N-protected peptideof formula N-Boc-Phe-Gly-Phe-Gly-OH, (217 mg) in a similar manner toExample 1, followed by the N-acetylation with acetic anhydride, wherebya complex of N-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe-Boc, (98 mg,with the content of the N-Boc-peptide of 28%) was prepared. This complex(80 mg) was deprotected by acid treatment for removal of the Boc groupsin a similar manner to Example 1, to afford a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe-H, (46 mg, with a peptidecontent of 24%).

The active ester of methotrexate (MTX, 69 mg) was reacted with 35 mg ofthe above complex in a similar manner to Example 1, so that a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Gly-Phe--MTX (35 mg, with an MTXcontent of 19%) was obtained. With respect to this complex, theMTX/peptide molar ratio in the complex and the value a/(a+b) defined forthe formula (I) were 0.92 and 0.19, respectively, as calculated inaccordance with the aforesaid equations (5) and (1).

EXAMPLE 11

The partially N-acetylated carboxymethylchitosan (50 mg), which had beenprepared in a similar manner to Example 10 and have a molecular weightof about 2×10⁴, was dissolved in a mixed solvent of water (1.25 ml) anddimethylsulfoxide (1.25 ml). In a similar manner to Example 1, an activeester of a protected peptide of formulaN-succinyl-Gly-Phe-Gly-Lys(ε-N-Boc)-O-tBu (169 mg) was reacted with thechitosan substance, whereby a complex ofpartially-N-acetylated-carboxymethylchitosan--Suc-Gly-Phe-Gly-Lys(.epsilon.-N-Boc)-O-tBuwas prepared. This complex was N-acetylated and then, deprotected byacid treatment to produce a complex ofN-acetylcarboxymethylchitosan--Suc-Gly-Phe-Gly-Lys-H, (47 mg).

The active ester of methotrexate (MTX) (23 mg) was reacted with 35 mg ofthe above complex, whereby a complex ofN-acetylcarboxymethylchitosan--Suc-Gly-Phe-Gly-Lys--MTX (29 mg, with anMTX content of 1.5 wt. %) was obtained. Ultraviolet-visible absorptionspectrum and GFC elution pattern of this complex are shown in FIG. 20and FIG. 21 of the accompanying drawings, respectively.

EXAMPLE 12

In a manner similar to Example 1, 25 mg of egg white lysozyme werereacted with 5.0 g of carboxymethylchitin (its carboxymethylation degreewas 0.7 per sugar residue) so that a depolymerized carboxymethylchitin(4.37 g) was obtained. 3.7 g portion of this chitin substance waspartially de-acetylated by alkali treatment, to give a partiallyN-acetylated carboxymethylchitosan (2.58 g) having a molecular weight ofabout 1×10⁵ (as measured using dextran as a standard substance).

The active ester (N-hydroxysuccinimido ester) of an N-protected peptideof formula N-Boc-Phe-Phe-Gly-OH, (141 mg) was reacted with theabove-mentioned partially N-acetylated carboxymethylchitosan (300 mg) ina similar manner to Example 1 to produce a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc, (340 mg).300 mg of this complex was N-acetylated with acetic anhydride so that acomplex of N-acetylcarboxymethylchitosan--Gly-Phe-Phe-Boc (298 mg, witha content of the N-Boc-peptide of 12.0%) was obtained.

A 100 mg portion of the above complex was deprotected by acid treatment(for removal of the Boc groups) in a similar manner to Example 1 toprepare a complex of N-acetylcarboxymethylchitosan--Gly-Phe-Phe-H, (89mg).

A 75 mg portion of this complex was dissolved in 1% aq. NaHCO₃ solution(7.5 ml), to which was then added the active ester of methotrexate (MTX)(75 mg). The reaction was made at 4° C. for 24 hours to link MTX to theN-terminals of the peptide moiety of the complex. The reaction solutionobtained was added to ethanol (40 ml). The resulting precipitate wascollected and then dried in vacuo, so that a complex ofN-acetyl-carboxymethylchitosan--Gly-Phe-Phe--MTX (75 mg, with an MTXcontent of 10.1 wt. %) was obtained as yellow powder.Ultraviolet-visible absorption spectrum and GFC elution pattern of thiscomplex are shown in FIG. 22 and FIG. 23 of the accompanying drawings,respectively.

EXAMPLE 13

2.0 g of carboxymethylchitin (its carboxymethylation degree was 0.7)were dissolved in 0.05M acetate buffered solution (pH 6.0, 200 ml), towhich egg white lysozyme (2.5 mg) was then added. The resulting solutionwas kept at 37° C. for 1.5 hours for the reaction, whereby adepolymerised carboxymethylchitin was obtained. This depolymerisedcarboxymethylchitin was then treated with aqueous 1N NaOH in a similarmanner to Example 1 to afford 1.72 g of partially N-acetylatedcarboxymethylchitosan having a molecular weight of about 3×10⁵ (asmeasured using dextran as a standard substance). This partiallyN-acetylated carboxymethylchitosan (150 mg) was dissolved in 0.5% aq.NaHCO₃ solution (15 ml) and then reacted with an active ester of anN-protected peptide of formula N-Boc-Ala-Gly-Gly-Gly-OH,(135 mg) in asimilar manner to Example 1, to prepare a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-Boc, (150 mg). 140 mgportion of this complex was N-acetylated with acetic anhydride toproduce a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-Boc,(154 mg). 130 mg portion of the latter complex was treated with acid toremove the Boc groups therefrom, so that a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-H (106 mg) was prepared.95 mg portion of this complex was dissolved in 1% aq. NaHCO₃ solution(4.8 ml) and then reacted with the active ester of MTX (86 mg) in theresulting solution, to produce a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala--MTX (88 mg, with an MTXcontent of 13.7 wt. %).

EXAMPLE 14

In a similar manner to Example 1, an active ester of an N-protectedpeptide of formula N-Boc-Gly-Gly-Gly-OH, (109 mg) was reacted with thepartially N-acetylated carboxymethylchitosan (150 mg) as obtained inExample 12, to prepare a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Gly-Boc, (167 mg).150 mg of this complex was N-acetylated with acetic anhydride, so that acomplex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Boc, (153 mg) wasprepared. A 130 mg portion of the latter complex was subjected to acidtreatment to remove the Boc groups therefrom and prepare a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-H, (110 mg). This complex(100 mg) was reacted with the active ester of MTX (90 mg), whereby acomplex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX (111 mg, withan MTX content of 17.4 wt. %) was obtained.

EXAMPLE 15

In a similar manner to Example 1, an active ester of an N-protectedpeptide of formula N-Boc-Ala-Gly-Gly-Gly-OH (135 mg) was reacted withthe partially N-acetylated carboxymethylchitosan (150 mg) as obtained inExample 12, to prepare a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Gly-Ala-Boc, (162mg). 150 mg of this complex was N-acetylated with acetic anhydride sothat a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-Boc,(157 mg) was produced. 130 mg of the latter complex was subjected toacid treatment to remove the Boc groups therefrom and prepare a complexof N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala-H, (114 mg). Thiscomplex (100 mg) was reacted with the active ester of MTX (90 mg),whereby a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-Ala--MTX(111 mg, with an MTX content of 17.4%) was obtained.

EXAMPLE 16

In a similar manner to Example 1, an active ester of an N-protectedpeptide of formula N-Boc-Gly-Phe-Gly-Gly-OH, (87 mg) was reacted withthe partially N-acetylated carboxymethylchitosan (200 mg) as obtained inExample 12, to prepare a complex of partiallyN-acetylated-carboxymethylchitosan--Gly-Gly-Phe-Gly-Boc, (206 mg). 100mg of this complex was N-acetylated with acetic anhydride so that acomplex of N-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly-Boc (92 mg,with a content of the N-Boc-peptide of 7.4%) was prepared. 80 mg portionof the latter complex was subjected to acid treatment to remove the Bocgroups therefrom and give a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly-H, (71 mg). This complex(50 mg) was reacted with the active ester of MTX (45 mg), whereby acomplex of N-acetylcarboxymethylchitosan--Gly-Gly-Phe-Gly--MTX complex(50 mg, with an MTX content of 7.0%) was obtained.

EXAMPLE 17

A 50 mg portion of the complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc (with itscarboxymethylation degree of 0.7 and a content of the N-Boc-peptide of13.2%), which was obtained in Example 12, was dissolved in 0.1% aq.NaHCO₃ solution (2.5 ml), to which was then added2-O-mono-methoxypolyethylene glycol-3,5-dichloro-s-triazine (80mg)(having a molecular weight of 5,000, a product of Sigma Corporation;hereinafter abbreviated as "PEG₁ "). The reaction was conducted at 0° C.for 4 hours. Water (2.5 ml), sodium hydrogen carbonate (500 mg) andacetic anhydride (200 μl) were added to the resulting reaction solutionwhich contained a produced complex ofpartially-N-acetylated-carboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-Boc,followed by reaction at 4° C. for 18 hours to complete the N-acetylationof said complex. The reaction solution obtained was dialyzed againstwater. The resultant non-dialysable solution was concentrated to 3 ml,to which acetone (90 ml) was added. The resulting precipitate wascollected, washed with methylene chloride and then dried in vacuo toobtain a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-Boc (72 mg, with thePEG content of 37%).

This complex (60 mg) was dissolved in 0.5 N HCl (3.5 ml) and theresulting solution was kept at 30° C. for 16 hours to effect thereaction for removal of the Boc groups from said complex. The reactionsolution obtained was added to a 1:2 mixed solvent (60 ml) of ethanoland ether. The resulting precipitate was collected, washed withmethylene chloride, and then dried in vacuo, so that a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-H (44 mg, with the PEGcontent of 26%) was obtained. A GFC elution pattern of this complex ispresented in FIG. 24 of the accompanying drawings. ThisN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-H complex (35 mg) wasdissolved in 1% aq. NaHCO₃ solution (1.75 ml), to which the active esterof MTX (16 mg) was added. The reaction was conducted at 4° C. for 18hours. The reaction solution obtained was dialyzed against water. Theresultant non-dialysable solution was concentrated to 3 ml, followed bythe addition of acetone (60 ml) thereto. The resulting precipitate wascollected, washed with methylene chloride and then dried in vacuo,whereby a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe--MTX (29 mg, with thePEG content of 21% and an MTX content of 7.9%) was obtained in the formof yellow powder as one example of the derivatives of the formula (X).Ultraviolet-visible absorption spectrum and GFC elution pattern of thelatter complex are shown in FIG. 25 and FIG. 26 of the accompanyingdrawings, respectively.

EXAMPLE 18

A 50 mg portion of thepartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc complex(with its carboxymethylation degree of 0.7 and a content of theN-Boc-peptide of 13.2%) which was obtained in Example 12 was dissolvedin 0.1% aq. NaHCO₃ solution (2.5 ml), and to the resulting solution wasadded the PEG₁ (12 mg) as used in Example 17. By effecting the reactionin a similar manner to Example 17, a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-Boc (49 mg, with thePEG content of 8.0%) was prepared. 40 mg of this complex was subjectedto the deprotecting treatment for removal of the Boc groups therefrom,to afford a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-H, (33 g). Thiscomplex (21 mg) was dissolved in 1% aq. NaHCO₃ solution (1.1. ml), towhich was then added the active ester of MTX (9.5 mg). The resultingmixture was kept at 4° C. for 18 hours for the reaction. The reactionsolution obtained was added to ethanol (11 ml). The resultingprecipitate was collected, washed with methylene chloride and then driedin vacuo, whereby a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe--MTX (17 mg, with thePEG content of 6.1% and an MTX content of 8.5%) was obtained.

EXAMPLE 19

A 50 mg portion of thepartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc complex(with its carboxymethylation degree of 0.7 and a content of theN-Boc-peptide of 14.8%), which had been prepared according to Example 12above, was dissolved in 0.1% aq. NaHCO₃ solution (5 ml), followed byaddition of PEG₁ (200 mg) to said solution. By effecting the reaction ina similar manner to Example 17, a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-Boc (100 mg, with thePEG content of 58%) was prepared. A 90 mg portion of the latter complexwas subjected to the acid treatment for removal of the Boc groups, togive a complex of N-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe-H(67 mg, with the PEG content of 45%). In a similar manner to Example 17,this complex (50 mg) was reacted with the active ester of MTX, whereby acomplex of N-acetylcarboxymethylchitosan-(PEG₁)--Gly-Phe-Phe--MTX (42mg, with the PEG content of 43% and an MTX content of 6.3%) wasobtained.

EXAMPLE 20

A 25 mg portion of a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Phe-Gly-Boc (withits carboxymethylation degree of 0.7 and a content of the N-Boc-peptideof 7.9%), which had been prepared in a similar manner to Example 16, wasdissolved in 0.1% aq. NaHCO₃ solution (1.25 ml), to which was added thePEG₁ (50 mg). By effecting the reaction in a similar manner to Example17, a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Gly-Phe-Gly-Boc (42 mg, withthe PEG content of 42%) was prepared. 30 mg of this complex wassubjected to the acid treatment for removal of the Boc groups, to afforda complex of N-acetylcarboxymethylchitosan-(PEG₁)--Gly-Gly-Phe-Gly-H,(20 mg). The latter complex was reacted with the active ester of MTX ina similar manner to Example 17, so that a complex ofN-acetylcarboxymethylchitosan-(PEG₁)--Gly-Gly-Phe-Gly--MTX (11 mg, withthe PEG content of 27% and an MTX content of 6.1%) was produced.

EXAMPLE 21

A 100 mg portion of the complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Boc (with itscarboxymethylation degree of 0.7 and a content of the N-Boc-peptide of14.8%), which had been prepared in a similar manner to Example 12, wasdissolved in 0.5% aq. NaHCO₃ solution (10 ml), to which was then addeddimethylformamide (8.5 ml) to give a uniform solution. To this solutionwas added an active ester (100 mg) of methoxypolyoxyethylene carboxylicacid (with a molecular weight of 5000, a product of Sigma Corporation;hereinafter abbreviated as "PEG_(co) "), and the resulting solution waskept at 4° C. for 18 hours to effect the reaction to combine thePEG_(co) chain with the non-N-acetylated amino groups which were presentin the sugar units of said complex. The resulting reaction solution wasdialyzed against water. Sodium hydrogen carbonate (3.6 g) and aceticanhydride (2.1 ml) were added to the resulting non-dialysable solution(27ml), followed by effecting the acetylation at 4° C. for 20 hours, sothat thepartially-N-acetylated-carboxymethylchitosan-(PEG_(co))--Gly-Phe-Phe-Boccomplex was N-acetylated. The reaction solution obtained was dialyzedagainst water. The resultant non-dialysable solution was concentrated to6 ml, to which acetone (120 ml) was added. The resulting precipitate wascollected, washed with methylene chloride and then dried in vacuo, toafford a complex of anN-acetylcarboxymethylchitosan-(PEG_(co))--Gly-Phe-Phe-Boc (109 mg, withthe PEG content of 9.0%). 50 mg portion of the latter complex wassubjected to the acid treatment for removal of the Boc groups therefrom,to prepare a complex ofN-acetylcarboxymethylchitosan-(PEG_(co))--Gly-Phe-Phe-H (35 mg, with thePEG content of 4.7%). This complex (25 mg) was reacted with the activeester of MTX in a similar manner to Example 18, whereby a complex ofN-acetylcarboxymethylchitosan-(PEG_(co))--Gly-Phe-Phe--MTX (25 mg, withthe PEG content of 4.5% and an MTX content of 9.1%) was produced.

EXAMPLE 22

With a partially N-acetylated carboxymethylchitosan (300 mg) which wasprepared as in Example 1, was reacted an active ester of an N-protectedpeptide of formula N-Boc-Gly-Gly-Gly-OH (289 mg) in a similar mammer toExample 1, to prepare apartially-N-acetylated-carboxymethylchitosan--Gly-Gly-Gly-Boc complex(334 mg).

A 125 mg portion of this complex was dissolved in water (12 ml), towhich was then added 3.3% aq. sodium meta-periodate (5 ml). The reactionwas effected at room temperature for 4 hours in the dark. After theresulting reaction solution was dialyzed against water, the resultingnon-dialysable solution was concentrated to about 12 ml, to which sodiumborohydride (30 mg) was added. The reduction reaction was then effectedovernight. The pH of the resulting reaction solution was lowered to 4.5and thereafter was raised back to 8.0 and then added into ethanol (60ml). The resulting precipitate was collected and then dried in vacuo,whereby there was prepared a complex ofN-acetylcarboxymethylchitosan--Gly-Gly-Gly-Boc (77 mg), of which somepyranose rings had been opened and which was converted into thederivative of a polyol.

A 60 mg portion of this complex was subjected to the acid treatment in asimilar manner to Example 1 to remove the Boc groups from the complex,whereby a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly-H asconverted into a polyol derivative was prepared (yield: 46 mg). Anactive ester of MTX (46 mg) was reacted with 35 mg of the latter complexto produce a complex of N-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTXas converted into a polyol derivative (42 mg, with an MTX content of29%). Ultraviolet-visible absorption spectrum and GFC elution pattern ofthe last-mentioned complex are shown in FIG. 27 and FIG. 28 of theaccompanying drawings respectively.

EXAMPLE 23

With a partially N-acetylated carboxymethylchitosan (250 mg) which wasprepared similarly to Example 1, was reacted an active ester of anN-protected peptide of formula N-Boc-Gly-Phe-Phe-Gly-OH (527 mg) in asimilar manner to Example 1, to prepare a complex ofpartially-N-acetylated-carboxymethylchitosan--Gly-Phe-Phe-Gly-Boc, (327mg). A 200 mg portion oft his complex was subjected to periodateoxidation and then reduced in a similar manner to Example 22 above.Thus, a complex of N-acetylcarboxymethylchitosan--Gly-Phe-Phe-Gly-Bocwhich had been converted into the polyol derivative (113 mg, with acontent of the N-Boc-peptide of 28%) was prepared.

A 60 mg portion of the latter complex was subjected to the acidtreatment in a similar manner to Example 1 to remove the Boc groups fromsaid complex. Thereby, a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Phe-Gly-H which was convertedinto the polyol derivative (36 mg, with a peptide content of 24%) wasprepared. This complex (30 mg) was reacted with the active ester of MTX(55 mg) to produce a complex ofN-acetylcarboxymethylchitosan--Gly-Phe-Phe-Gly--MTX (22 mg, with an MTXcontent of 19%) which was converted into the polyol derivative. Withrespect to this complex, the MTX/peptide molar ratio in said complex andits value of a/(a+b) as defined for the formula (I) were 0.93 and 0.19as calculated by the aforesaid equations (5) and (1), respectively.

Properties of the derivatives having the formula (I), (X) or (XI)according to this invention were tested by the following Experiments1-4.

EXPERIMENT 1 Samples and Test Procedures

The N-acetylcarboxymethylchitosan--Gly-Phe-Phe--MTX complex obtained inExample 1, as well as theN-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex obtained inExample 2 were taken as test samples and individually dissolved inphysiological saline to prepare two solutions of 400 μg/ml of thesample. To plasma (190 μl) which had been obtained by centrifuging avolume of blood collected from several mice was added a 10 μl portion ofeach solution of the sample, followed by reaction at 37° C. From theresulting reaction mixture were separated test solutions at intervals oftime. After each test solution of the reaction mixture wasdeproteinized, the test solutions were analyzed by gel filtrationchromatographic method (using a column of TSK-gel G4000 PW_(XL), aseluted with 0.1M NaCl at flow rate of 0.8 ml/min. and at columntemperature of 40° C., while the detection being made by ultravioletabsorption (at 307 nm), so that the percentages of the residual contentof MTX, which retained the initial form of its complex within the plasmawere evalutated.

Test Results

The test results are plotted in FIG. 16 of the accompanying drawings,which is a graph showing time-dependent variations in the percentages ofthe residual content of MTX, which was present in the plasma withretaining the form of the complex represented by the formula (I). Inthis graph, the broken line with dots () indicates the test resultsobtained with the N-acetylcarboxymethylchitosan--Gly-Phe-Phe--MTXcomplex, while the full line with squares (□) indicates the test resultsobtained with the N-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTXcomplex.

It is observed from FIG. 16 that the above two complexes are practicallyfree from their degradation in the blood circulation and remain stablein the blood.

EXPERIMENT 2 Samples and Test Procedures

The N-acetylcarboxymethylchitosan--Gly-Phe-Phe--MTX complex (1 mg)obtained in Example 1, as well as theN-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex obtained inExample 2 were taken as test samples and individually reacted in 0.1Macetate buffer (pH 6.0, 1 ml) at 37° C. in the presence or absence ofegg white lysozyme (10 μg). From the reaction mixture at the ends of 1,3, 6 and 23 hours were taken test solutions, which were then analyzed bygel filtration chromatographic method as described in Experiment 1. Inthis way, prolongation in the elution time of the complex (upon the gelfiltration) as involved by decrease in the molecular weight of thecomplex dut to the degradation of its N-acetylcarboxymethylchitosanmoiety was evaluated.

Test Results

The test results are plotted in FIG. 17 of the accompanying drawings,which is a graph showing how the reaciton time was co-related to theelution time at which the elution of a peak of the complex appeared uponthe elution of the reaction mixture at the different points of thereaction time. In the graph, the line with circles (∘) and the brokenline with dots () indicate respectively the test results obtained withthe N-acetylcarboxymethylchitosan--Gly-Phe-Phe--MTX complex tested inthe absence of lysozyme and tested in the presence of lysozyme. While,the line with squares (□) and the broken line with solid squares ()indicate respectively the test results obtained with theN-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex tested in theabsence of lysozyme and tested in the presence of lysozyme.

It is observed from FIG. 17 that the N-acetylcarboxymethylchitosanmoiety of the tested complexes undergone the degradation under theaction of lysozyme and, as a result, the complexes were both decreasedin their molecular weight. Therefore, it is expected that the complexescannot remain for a too long time in the body.

EXPERIMENT 3 Samples

The N-acetylcarboxymethylchitosan--Gly-Gly-Gly--MTX complex of Example2, in which the MTX portion had been ³ H-labeled, was used as a testsample compound. In addition, an ³ H-labeled product of the partiallyN-acetylated carboxymethylchitosan as obtained in the course of theprocess of Example 1 was employed as a referential sample.

Test Procedures

In the following experiment, there were employed tumor-bearing male ICRmice which had received subcutaneous implantation of Sarcoma 180 andsubsequently had been raised for 10 days. The test sample compound wasdissolved in physiological saline. Using three mice per group, thesolution of the test sample compound was administered at a dose of 20mg/Kg through the caudal vein. At the ends of 15 minutes, 30 minutes, 1hour, 2 hours, 6 hours and 24 hours after the administration, thefemoral artery and femoral vein of the mice of the respective groupswere cut to collect blood samples. The radioactivity of a serum sample,which had been obtained by centrifuging each of the blood samples, andthe radioactivity of a cancer tissue taken from the corresponding mousewere measured by the combustion method. In this way, the concentrationsof the test sample compound present in the serum and the cancer tissuewere evaluated.

Test Results

The test results are plotted in FIGS. 18 and 19 of the accompanyingdrawings. FIG. 18 is a graph showing time-dependent variations in theconcentration of the test sample compound present in the blood serum,whereas FIG. 19 is a graph showing time-dependent variations in theconcentration of the test sample compound present in the cancer tissue.In both of the graphs, the line with squares (□) show the resultsobtained with the referential sample, whereas the line with dots () showthe results obtained with the test sample complex.

It is understood from FIG. 18 that the test sample complex would beexpected to promptly disappear in the blood circulation because it wasin the form of a complex having MTX linked to the polysaccharide(N-acetylcarboxymethylchitosan) via the peptide chain of the complex,but that, contrary to the expectation, however, the retention in bloodof the test sample compound having the form of the complex is actuallycomparable to or longer than the retention of the simple polysaccharidewhich was used as the referential sample having no MTX and peptide chainthereto, and which is namely the N-acetylcarboxymethylchitosan itself.It is also observed from FIG. 19 that, as compared with theabove-mentioned simple polysaccharide, the test sample complex can benewly imparted with such property that the complex can concentrate in acancer tissue owing to the possession of the peptide chain by thecomplex. Therefore, it is feasible that the substance of the formula (I)according to this invention gains the tendency of organotropism byselection of a suitable peptide chain (--X--) to be contained in thesubstance of the formula (I) according to this invention.

EXPERIMENT 4

In this Experiment, the following experiments were conducted usingseveral examples of the invention substances of the formula (X) as testsamples.

Thus, the complexes which were obtained as the final products ofExamples 17, 18 and 19 above and had the polyethylene glycol (PEG)contents of 6%, 21% and 43%, respectively, and of which the MTX portionswere labelled with ³ H, were prepared and employed as the ³ H-labeledtest compounds for use as Test Samples 1, 2 and 3. Further, thecomplexes same as those above-mentioned complexes except that the PEGportion was eliminated therefrom and the MTX portion was ³ H-labeled,were also prepared and used as referential ³ H-labeled compounds. Ineach of the complexes used as Test Samples 1, 2 and 3 and the complexesused as the referential complexes, the peptide chain which was bonded tothe 2-amino group of the carboxymethylglucosamine unit of these testedcomplexes was Gly-Phe-Phe. The PEG portion present in the complexes asTest Samples 1, 2 and 3 was the PEG₁ (molecular weight: 5,000) asdescribed in Example 17.

Test Procedures

In the following experiment, there were employed such tumor-bearingfemale Wistar rats which had received the subcutaneous transplantationof cancer cells, Walker 256, to an inguinal part and subsequently hadbeen raised for 6 days. Each solution of the test sample was preparedfor administration, by suitably diluting the ³ H-labeled test samplecomplex with a solution of a corresponding non-radioactively labeledcomplex in physiological saline. Using three rats per group, thesolution of the test sample was administered into the carotids of ratsat a dose of 10 mg/Kg of the complex.

Under ether anesthesia, blood was sampled from the carotid of ratsperiodically at the ends of 30 minutes, 1 hour, 2 hours, 4 hours and 6hours after the administration, and the blood was centrifuged toseparate the plasma. Upon elapsed time of 24 hours after theadministration, blood was collected from rats under ether enesthesiauntil the rats were sacrificed under exsanguination. Several tissues andorgans were separated from the sacrificed rats. After weighing the wholeor some parts of the tissues and organs, their radioactivities weremeasured.

The radioactivity of the tissues separated was measured in the followingmanner. After each sample of tissue was collected in a combustion coneand was dried, the sample was combusted in an automaticsample-combusting apparatus ("ASC-113", ALOKA). The resulting ³ H₂ O wasadded to a scintillator ("AQUASOL-2", NEN) and then measured by liquidscintillation counter ("LSC-3600", ALOKA). The value of radioactivity someasured was calibrated by the external standard radiation sourcemethod.

Test results

The test results are presented in FIG. 29 and FIG. 30 of theaccompanying drawings as well as in Table 1 given hereinafter. FIG. 29is a graph showing time-dependent variations in the concentration ofeach sample complex present in the plasma as measured up to the end of24 hours after the administration of the complex. FIG. 30 is a graphshowing the distributions of each sample complex present in tissues ofthe rat body which are expressed as the concentration of the complex interm of the percentages on the basis of the dose of the complex. Table 1shows the AUC value of each sample complex as evaluated on the basis ofthe graph of FIG. 29 and also shows the concentration of each samplecomplex in the tumor as expressed in percentages relative to the dose ofthe complex.

As is indicated by FIG. 29, the tested complex samples exhibits that thecomplex has a better retention in the blood circulation as theproportion of the PEG introduced in the complex becomes greater. Whenthis is reviewed with reference to the AUC values of Table 1, it isunderstood that, as compared with the referential complexes, theretention of the Test Sample complexes in the blood was increased muchby about 1.3 times with Test Sample 1 (having the introduced PEG contentof 6%) and by about 3 times with Test Sample 2 (having the introducedPEG content of 21%) and also with Test Sample 3 (having the introducedPEG content of 43%).

As is envisaged from FIG. 30, it is also recognized that, in regard tothe tumor tissue, the concentrations of the tested complexes present inthe tumor can steadily increase with the higher content of theintroduced PEG of the complexes, whereas in regard to the organs otherthan the tumor tissue, the concentrations of the tested complexes inthese organs are not distinctly different from each other, depending onthe content of the introduced PEG of the complexes. Specifically, thisis as revealed from the values of the concentrations of the testedcomplexes in the tumor shown in Table 1. Thus, the introduction of thePEG moiety into an N-acetylcarboxymethylchitosan derivative can resultin that the complex gains appearance of a tendency of the complex torecognize a cancer tissue. Therefore, use of such complex having an PEGcontent makes it possible to reduce the required dose of an anticanceragent and hence to reduce undersirable effects of the anticancer agenton other organs.

                  TABLE 1                                                         ______________________________________                                                                Concentration of Sample                                         AUC.sub.o-oo in Plasma                                                                      in Tumor (% of dose/g at                              Sample    (% of dose · hr/ml)                                                                end of 24 hrs)                                        ______________________________________                                        Referential                                                                             15.83         0.18                                                  Sample                                                                        Test Sample 1                                                                           20.85         0.22                                                  Test Sample 2                                                                           46.20         0.35                                                  Test Sample 3                                                                           45.52         0.44                                                  ______________________________________                                    

Industrial Utility of the Invention

The novel N-acetylcarboxymethylchitosan derivatives according to thisinvention are intended to achieve the in vivo targeting ofpharmaceutical compounds. The novel derivatives of this invention areuseful as high-molecular carriers of polysaccharide-type which arecapable of enhancing the stability of such pharmaceutical compounds inblood, the organotropism of such pharmaceutical compounds and thebiodegradability of such pharmaceutical products. Further, the novelN-acetylcarboxymethylchitosan derivatives of this invention as providedin the form of their complexes having linked to pharmaceutical compoundshave organotropism and have also other advantageous properties and henceare useful as pharmaceutical products.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 20                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE: N-terminal                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyPheGlyGly                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PheGlyPheGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 ( xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                      GlyPheGlyPhe                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaGlyGlyGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AlaAlaValAla                                                                 1                                                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyGly GlyValAla                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GlyGlyGlyLeuAla                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 ( xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      GlyGlyPheLeuGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyGlyPheTyrAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyGlyGlyGlyGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlyGlyGlyPheLeuGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlyGlyGlyGlyLeuAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GlyGlyGlyGlyGlyGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyGlyGlyGlyPheLeuGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyGlyGlyGlyGlyGlyGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyGlyGlyGlyGlyLeuAla                                                         15                                                                            (2 ) INFORMATION FOR SEQ ID NO:17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlyGlyGlyGlyGlyPheLeuGly                                                       15                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyGlyGlyGly GlyGlyGlyGly                                                     15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GlyGlyGlyGlyGlyGlyGlyGlyGly                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE: N-terminal                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlyGlyGlyGlyGlyGlyGlyGlyGlyGly                                                1510                                                                      

We claim:
 1. An N-acetylcarboxymethylchitosan derivative represented bythe following formula (Ia): ##STR17## wherein R₁ and R₂ individuallymean H or carboxymethyl group with the proviso that R₁ and R₂ do notstand for H at the same time, P denotes such residue of methotrexatewhich is formed by removal of an --OH group from any one of the twocarboxyl groups of methotrexate as represented by the formula: ##STR18##and Q stands for H, X represents a peptide chain having the formula-Gly-Gly-Gly- and terminated by --NH-- towards P or Q; a₁ represents apositive integer and a₂ represents zero, and b stands for a positiveinteger; and wherein a₁ and b are selected so that saidN-acetylcarboxymethylchitosan derivative has the followingcharacteristic values (1)-(4):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                       1.0                                                 (2)    molecular weight (as measured                                                                    1 × 10.sup.5                                         by gel filtration method):                                             (3)    a/(a + b):         0.2                                                        [provided that a = a.sub.1 + a.sub.2 ]                                 (4)    P/X ratio (molar ratio):                                                                          1.0.                                               ______________________________________                                    


2. An N-acetylcarboxymethylchitosan derivative represented by thefollowing formula (Ib): ##STR19## wherein R₁ and R₂ individually mean Hor carboxymethyl group; with proviso that R₁ and R₂ do not stand for Hat the same time,Q stands for H or a group --OH, X represents a chain ofthe formula

    -(Suc)q--X'--

where Suc means the succinic acid residue of the formula --OC--CH₂ --CH₂--CO--, q is an integer of zero or 1 and X' means a peptide chaincontaining two to six amino acids such that when q is zero, said peptidechain --X'-- is terminated by --NH-- towards Q and Q is H, and such thatwhen q is 1, said peptide chain --X'-- is terminated by --CO-- towards Qand Q is --OH; a₂ represents a positive integer, and b stands for apositive integer; and wherein a₂ and b are selected so that saidN-acetylcarboxymethylchitosan derivative has the followingcharacteristic values (1)-(3):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a.sub.2 /(a.sub.2 + b):                                                                            0.01-1.                                           ______________________________________                                    


3. An N-acetylcarboxymethylchitosan derivative represented by thefollowing formula (Ib-1): ##STR20## wherein R₁ and R₂ individually meanH or carboxymethyl group; with the proviso that R₁ and R₂ do not standfor H at the same time,Q stands for H or a group --OH, X represents apeptide chain containing two to six amino acids and terminated by --NH--towards Q where Q is H, or X represents a chain of the formula

    -(Suc)--X--

where Suc means the succinic acid residue of the formula --OC--CH₂ --CH₂--CO-- and X' means a peptide chain containing two to six amino acidsand terminated by --CO-- towards Q where Q is --OH, a₂ represents apositive integer, and b stands for a positive integer; and wherein a₂and b are selected so that said N-acetylcarboxymethylchitosan derivativehas the following characteristic values (1)-(3):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by filtration method):                                                 (3)    a.sub.2 /(a.sub.2 + b):                                                                            0.01-1.                                           ______________________________________                                    


4. An N-acetylcarboxymethylchitosan derivative represented by thefollowing formula (Ic): ##STR21## wherein R₁ and R₂ individually mean Hor carboxymethyl group; with the proviso that R₁ and R₂ do not stand forH at the same time,X represents a peptide chain containing two to sixamino acids and terminated by --NH-- towards P' or Q, where P' denotesan alkoxycarbonyl group or an aralkyloxycarbonyl group serving as anamino-protecting group and where Q stands for H, or X represents a chainof the formula

    -(Suc)--X'--

where Suc means the succinic acid residue of the formula --OC--CH₂ --CH₂--CO-- and X' means a peptide chain containing two to six amino acidsand terminated by --CO-- towards P' or Q, where P' denotes a loweralkoxyl group or an aralkyloxy group or a lower alkylimino group servingas a carboxyl-protecting group and where Q stands for a group --OH, a₁and a₂ individually represent a positive integer, and b stands for apositive integer; and wherein a₁, a₂ and b are selected so that saidN-acetylcarboxymethylchitosan derivative has the followingcharacteristic values (1)-(4):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a/(a + b):           0.01-1                                                   [provided that a = a.sub.1 + a.sub.2 ]                                 (4)    P'X ratio (molar ratio):                                                                           0.01-1.                                           ______________________________________                                    


5. An N-acetylcarboxymethylchitosan derivative according to claim 4,wherein X present in XP' means a peptide chain composed of 1-4 aminoacids and P' is bonded to an --NH-terminated amino acid of said peptidechain.
 6. An N-acetylcarboxymethylchitosan derivative according to claim4, wherein XP' means one of the following:P'-Phe-Phe-Gly-,P'-Gly-Phe-Gly-Gly- (SEQ ID NO:1), P'-Phe-Gly-Phe-Gly- (SEQ ID NO:2),P'-Gly-Phe-Gly-Phe- (SEQ ID NO:3), P'-Gly-Gly-Gly-, orP'-Ala-Gly-Gly-Gly- (SEQ ID NO:4),and P' means an alkoxycarbonyl groupor an aralkyloxycarbonyl group serving as the amino-protecting groupwhich is bonded to the --NH-terminated amino acid of the peptide chain.7. An N-acetylcarboxymethylchitosan derivative according to claim 4,wherein X' means a peptide chain composed of 1-4 amino acids and P' isbonded to a --CO-terminated amino acid of said peptide chain.
 8. AnN-acetylcarboxymethylchitosan derivative according to claim 4, whereinXP' is selected from the group consisting of:-(Suc)-Ala-Ala-Ala-P' and-(Suc)-Ala-Ala-Val-Ala-P' (SEQ ID NO:5)in which Suc represents thesuccinic acid residue and P' means a lower alkoxy group serving as acarboxyl-protecting group which is bonded to the --CO-terminated aminoacid of the peptide chain.
 9. An --N-acetylcarboxymethylchitosanderivative according to claim 4, wherein P' is a tert-butoxycarbonylgroup, a p-methoxybenzyloxycarbonyl group, or a tertbutyloxy group. 10.A partially N-acetylated carboxymethylchitosan derivative represented bythe following formula (IIa): ##STR22## wherein R₁ and R₂ individuallymean H or carboxymethyl group; with the proviso that R₁ and R₂ do notstand for H at the same time,X represents a peptide chain containing twoto six amino acids and terminated by --NH-- towards P", where P" denotesa hydrogen atom, an alkoxycarbonyl group or" an aralkyloxycarbonyl groupserving as an amino-protecting group, or X represents a chain of theformula

    --(Suc)--X'--

where Suc means the succinic acid residue of the formula --O--CH₂ --CH₂--CO-- and X' means a peptide chain containing two to six amino acidsand terminated by --CO-- towards P" where P" denotes a hydroxyl group, alower alkoxyl group or an aralkyloxy group or a lower alkylimino groupserving as a carboxyl-protecting group, and a, b₁ and b₂ individuallyrepresent a positive integer; and wherein a, b₁ and b₂ are selected sothat said N-acetylated carboxymethylchitosan derivative has thefollowing characteristic values (1)-(4):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a/(a + b):           0.01-1                                                   [provided that b = b.sub.1 + b.sub.2 ]                                 (4)    P"/X ratio (molar ratio):                                                                          0.1-1.                                            ______________________________________                                    


11. An N-acetylcarboxymethylchitosan derivative represented by thefollowing formula (Xa) or (XIa): ##STR23## wherein R₁ and R₂individually mean H or carboxymethyl group with the proviso that R₁ andR₂ do not stand for H at the same time,X represents a peptide chaincontaining two to six amino acids and terminated by --NH-- towards Q,where Q stands for H, or X represents a chain of the formula

    --(Suc)--X'--

where Suc means the succinic acid residue of the formula --OC--CH₂ --Ch₂--CO-- and X' means a peptide chain containing two to six amino acidsand terminated by --CO-- towards Q, where Q stands for a group --OH, a₂represents a positive integer, and b and c individually stand for apositive integer, and -PEG represents a group having the followingformula (VI), (VII), (VIII) or (IX): ##STR24##

    --CO--CH.sub.2 CH.sub.2 --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (VII)

    --CO--O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3            (VIII)

    --CO--CH.sub.2 --O--(CH.sub.2 CH.sub.2 O).sub.n --CH.sub.3 (IX)

in which n means the average polymerization degree of the polyethyleneglycol chain; and wherein a₂, b and c are selected so that saidN-acetylcarboxymethylchitosan derivative has the followingcharacteristic values (1)-(3):

    ______________________________________                                        (1)    carboxymethylation degree:                                                                         0.5-1.2                                           (2)    molecular weight (as measured                                                                      3,000-300,000                                            by gel filtration method):                                             (3)    a.sub.2 /(a.sub.2 + b + c):                                                                        0.01-1.                                           ______________________________________                                    